Derivatized oligonucleotides having improved uptake and other properties

ABSTRACT

Linked nucleosides having at least one functionalized nucleoside that bears a substituent such as a steroid molecule, a reporter molecule, a non-aromatic lipophilic molecule, a reporter enzyme, a peptide, a protein, a water soluble vitamin, a lipid soluble vitamin, an RNA cleaving complex, a metal chelator, a porphyrin, an alkylator, a pyrene, a hybrid photonuclease/intercalator, or an aryl azide photo-crosslinking agent exhibit increased cellular uptake and other properties. The substituent can be attached at the 2′-position of the functionalized nucleoside via a linking group. If at least a portion of the remaining liked nucleosides are 2′-deoxy-2′-fluoro, 2′-O-methoxy, 2′-O-ethoxy, 2′-O-propoxy, 2′-O-aminoalkoxy or 2′-O-allyloxy nucleosides, the substituent can be attached via a linking group at any of the 3′ or the 5′ positions of the nucleoside or on the heterocyclic base of the nucleoside or on the inter-nucleotide linkage linking the nucleoside to an adjacent nucleoside.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.782,374, filed Oct. 24, 1991, which is a continuation-in-part ofPCT/US91/00243, filed Jan. 11, 1991, which is a continuation-in-part ofapplication Ser. No. 463,358, filed Jan. 11, 1990, and of applicationSer. No. 566,977, filed Aug. 13, 1990. The entire disclosures of each ofthese applications, which are assigned to the assignee of thisapplication, are incorporated herein by reference.

FIELD OF THE INVENTION

This application is directed to sequence specific oligonucleotides thatinclude functionalized nucleosides having substituents such as steroids,reporter molecules, reporter enzymes, non-aromatic lipophilic molecules,peptides, or proteins attached via linking groups.

BACKGROUND OF THE INVENTION

Messenger RNA (mRNA) directs protein synthesis. Antisense methodology isthe complementary hybridization of relatively short oligonucleotides tomRNA or DNA such that the normal, essential functions of theseintracellular nucleic acids are disrupted. Hybridization is thesequence-specific hydrogen bonding of oligonucleotides to RNA orsingle-stranded DNA via complementary Watson-Crick base pairs.

The naturally occurring events that provide the disruption of thenucleic acid function, discussed by Cohen in Oligonucleotides: AntisenseInhibitors of Gene Expression, CRC Press, Inc., Boca Raton, Fla. (1989),are thought to be of two types. The first, hybridization arrest, denotesa terminating event in which the oligonucleotide inhibitor binds to thetarget nucleic acid and thus prevents, by simple steric hindrance, thebinding of essential proteins, most often ribosomes, to the nucleicacid. Methyl phosphonate oligonucleotides (see, e.g., Miller, et al.,Anti-Cancer Drug Design 1987, 2, 117) and α-anomer oligonucleotides, thetwo most extensively studied antisense agents, are thought to disruptnucleic acid function by hybridization arrest.

The second type of terminating event for antisense oligonucleotidesinvolves the enzymatic cleavage of targeted RNA by intracellular RNaseH. A 2′-deoxyribofuranosyl oligonucleotide or oligonucleotide analoghybridizes with the targeted RNA to form a duplex that activates theRNase H enzyme to cleave the RNA strand, thus destroying the normalfunction of the RNA. Phosphorothioate oligonucleotides provide the mostprominent example of antisense agents that operate by this type ofantisense terminating event.

Considerable research is being directed to the application ofoligonucleotides and oligonucleotide analogs as antisense agents fordiagnostics, research reagents, and therapeutics. At least fortherapeutic purposes, the antisense oligonucleotides and oligonucleotideanalogs must be transported across cell membranes or taken up by cellsto express activity. One method for increasing membrane or cellulartransport is by the attachment of a pendant lipophilic group.

Ramirez, et al., J. Am. Chem. Soc. 1982, 104:, 5483, introduced thephospholipid group 5′-O-(1,2-di-O-myristoyl-sn-glycero-3-phosphoryl)into the dimer TpT independently at the 3′ and 5′ positions.Subsequently Shea, et al., Nuc. Acids Res. 1990, 18, 3777, disclosedoligonucleotides having a 1,2-di-O-hexyldecyl-rac-glycerol group linkedto a 5′-phosphate on the 5′-terminus of the oligonucleotide. Certain ofthe Shea, et. al. authors disclosed these and other compounds in patentapplication PCT/US90/01002. Another glucosyl phospholipid was disclosedby Guerra, et al., Tetrahedron Letters 1987, 28, 3581.

In other work, a cholesteryl group was attached to the inter-nucleotidelinkage between the first and second nucleotides (from the 3′ terminus)of an oligonucleotide. This work is disclosed in U.S. Pat. No. 4,958,013and by Letsinger, et al., Proc. Natl. Acad. Sci. USA 1989, 86, 6553. Thearomatic intercalating agent anthraquinone was attached to the 2′position of a sugar fragment of an oligonucleotide as reported byYamana, et al., Bioconjugate Chem. 1990, 1, 319.

Lemairte, et al., Proc. Natl. Acad. Sci. USA 1986, 84, 648 and Leonetti,et al., Bioconjugate Chem. 1990, 1, 149, disclose modifying the 3′terminus of an oligonucleotide to include a 3′-terminal ribose sugarmoiety. Poly(L-lysine) was linked to the oligonucleotide via periodateoxidation of this terminal ribose followed by reduction and couplingthrough a N-morpholine ring. Oligonucleotide-poly(L-lysine) conjugatesare described in European Patent application 87109348.0, wherein thelysine residue was coupled to a 5′ or 3′ phosphate of the 5′ or 3′terminal nucleotide of the oligonucleotide. A disulfide linkage has alsobeen utilized at the 3′ terminus of an oligonucleotide to link a peptideto the oligonucleotide, as described by Corey, et al., Science 1987,238, 1401; Zuckermann, et al., J. Am. Chem. Soc. 1988, 110, 1614; andCorey, et al., J. Am. Chem. Soc. 1989, 111, 8524.

Nelson, et al., Nuc. Acids Res. 1989, 17, 7187 describe a linkingreagent for attaching biotin to the 3′-terminus of an oligonucleotide.This reagent, N-Fmoc-O-DMT-3-amino-1,2-propanediol, is commerciallyavailable from Clontech Laboratories (Palo Alto, Calif.) under the name3′-Amine on and from Glen Research Corporation (Sterling, Va.) under thename 3′-Amino-Modifier. This reagent was also utilized to link a peptideto an oligonucleotide, as reported by Judy, et al., Tetrahedron Letters1992, 32, 879. A similar commercial reagent (actually a series oflinkers having various lengths of polymethylene connectors) for linkingto the 5′-terminus of an oligonucleotide is 5′-Amino-Modifier C6, alsofrom Glen Research Corporation. These compounds or similar ones wereutilized by Krieg, et al., Antisense Research and Development 1991, 1,161 to link fluorescein to the 5′-terminus of an oligonucleotide. Othercompounds of interest have also been linked to the 3′-terminus of anoligonucleotide. Asseline, et al., Proc. Natl. Acad. Sci. USA 1984, 81,3297 described linking acridine on the 3′-terminal phosphate group of anpoly (Tp) oligonucleotide via a polymethylene linkage. Haralambidis, etal., Tetrahedron Letters 1987, 28, 5199 reported building a peptide on asolid state support and then linking an oligonucleotide to that peptidevia the 3′ hydroxyl group of the 3′ terminal nucleotide of theoligonucleotide. Chollet, Nucleosides & Nucleotides 1990, 9, 957attached an Aminolink 2 (Applied Biosystems, Foster City, Calif.) to the5′ terminal phosphate of an oligonucleotide. They then used thebifunctional linking group SMPB (Pierce Chemical Co., Rockford, Ill.) tolink an interleukin protein to the oligonucleotide.

An EDTA iron complex has been linked to the 5 position of a pyrimidinenucleoside as reported by Dreyer, et al., Proc. Natl. Acad. Sci. USA1985, 82, 968. Fluorescein has been linked to an oligonucleotide in thesame manner, as reported by Haralambidis, et al., Nucleic Acid Research1987, 15, 4857 and biotin in the same manner as described in PCTapplication PCT/US/02198. Fluorescein, biotin and pyrene were alsolinked in the same manner as reported by Telser, et al., J. Am. Chem.Soc. 1989, 111, 6966. A commercial reagent, Amino-Modifier-dT from GlenResearch Corporation, can be utilized to introduce pyrimidinenucleotides bearing similar linking groups into oligonucleotides.

Cholic acid linked to EDTA for use in radioscintigraphic imaging studieswas reported by Betebenner, et al., Bioconjugate Chem. 1991, 2, 117;however, it is not known to link cholic acid to nucleosides, nucleotidesor oligonucleotides.

OBJECTS OF THE INVENTION

It is an object of this invention to provide sequence-specificoligonucleotides having improved transfer across cellular membranes.

It is a further object of this invention to provide improvements inresearch and diagnostic methods and materials for assaying bodily statesin animals, especially disease states.

It is an additional object of this invention to provide therapeutic andresearch materials having improved transfer and uptake properties forthe treatment of diseases through modulation of the activity of DNA orRNA.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with these and other objects evident from thisspecification, there are provided compounds that comprise a plurality oflinked nucleosides wherein at least one of the nucleosides isfunctionalized at the 2′-position with a substituent such as, forexample, a steroid molecule, a reporter molecule, a non-aromaticlipophilic molecule, a reporter enzyme, a peptide, a protein, a watersoluble vitamin, a lipid soluble vitamin, an RNA cleaving complex, ametal chelator, a porphyrin, an alkylator, a hybridphotonuclease/intercalator, a pyrene, or an aryl azidephoto-crosslinking agent. Preferably, the substituent is connected to2′-position using an intervening linking group.

In certain preferred embodiments of the invention, the substituentscomprise a steroid molecule, biotin, a reporter enzyme or a fluoresceindye molecule. In these embodiments, the steroid molecule is selectedfrom the group consisting of cholic acid, deoxycholic acid,dehydrocholic acid, cortisone, testosterone, cholesterol and digoxigeninwith the most preferred steroid molecule being cholic acid. Preferredreporter enzymes include horseradish peroxidase and alkalinephosphatase.

In further preferred embodiments, the non-aromatic lipophilic moleculeattached to the 2′-position comprises an alicyclic hydrocarbon,saturated or unsaturated fatty acid, wax, terpenoid, or polyalicyclichydrocarbon, including adamantane and buckminsterfullerenes. Waxesaccording to the invention include monohydric alcohol esters of fattyacids and fatty diamides. Buckminsterfullerenes include soccerball-shaped, cage molecules comprising varying numbers of covalentlybound carbon atoms. Terpenoids include the C₁₀ terpenes, C₂₀sesquiterpenes, C₃₀ diterpenes including vitamin A (retinol), retinoicacid, retinal and dehydroretinol, C₃₀ triterpenes, C₄₀ tetraterpenes andother higher polyterpenoids.

In other preferred embodiments, peptides or proteins attached to the2′-position comprise sequence-specific peptides and sequence-specificproteins, including phosphatases, peroxidases and nucleases.

Preferred linking molecules of the invention comprise Ω-aminoalkoxylinkers, Ω-aminoalkylamino linkers, heterobifunctional linkers orhomobifunctional linkers. A particularly preferred linking molecule ofthe invention is a 5-aminopentoxy group.

In preferred embodiments of the invention at least a portion of thelinked nucleosides are 2′-deoxy-2′-fluoro, 2′-methoxy, 2′-ethoxy,2′-propoxy, 2′-aminoalkoxy or 2′-allyloxy nucleosides. In otherpreferred embodiments of the invention the linked nucleosides are linkedwith phosphorothioate linking groups.

The invention also provides compounds that have a plurality of linkednucleosides. In preferred embodiments, at least one of the nucleosidesis: (1) a 2′-functionalized nucleoside having cholic acid linked to its2′-position; (2) a heterocyclic base functionalized nucleoside havingcholic acid linked to its heterocyclic base; (3) a 5′ terminalnucleoside having cholic acid linked to its 5′-position; (4) a 3′terminal nucleoside having cholic acid linked to its 3′-position; or (5)an inter-strand nucleoside having cholic acid linked to an inter-standlinkage linking said inter-strand nucleoside to an adjacent nucleoside.

In certain embodiments of the invention having linked nucleosides, atleast one linked nucleosides bears a 2′-deoxy′-2′-fluoro,2′-O—C₁-C₂₀-alkyl, 2′-O—C₂-C₂₀-alkenyl, 2′-O—C₂-C₂₀-alkynyl,2′-S—C₁-C₂₀-alkyl, 2′-S—C₂-C₂₀-alkenyl, 2′-S—C₂-C₂₀-alkynyl,2′-NH—C₁-C₂₀-alkyl, 2′-NH—C₂-C₂₀-alkenyl, or 2′-NH—C₂-C₂₀-alkynylsubstituent.

Further in accordance with the invention there is provided a method ofincreasing cellular uptake of a compound having a plurality of linkednucleosides that includes contacting an organism with a compound wherethe compound includes at least one nucleoside functionalized at the2′-position with a steroid molecule, a reporter molecule, a non-aromaticlipophilic molecule, a reporter enzyme, a peptide, a protein, a watersoluble vitamin, and a lipid soluble vitamin. The compound can beincluded in a composition that further includes an inert carrier for thecompound.

The invention also provides a method for enhancing the binding affinityand/or stability of an antisense oligonucleotide comprisingfunctionalizing the oligonucleotide with a steroid molecule, a reportermolecule, a non-aromatic lipophilic molecule, a reporter enzyme, apeptide, a protein, a water soluble vitamin, and a lipid solublevitamin.

DETAILED DESCRIPTION OF THE INVENTION

Antisense therapeutics can be practiced in a variety of organismsranging from unicellular prokaryotic and eukaryotic organisms tomulticellular eukaryotic organisms. Any organism that utilizes DNA-RNAtranscription or RNA-protein translation as a fundamental part of itshereditary, metabolic or cellular control is susceptible to antisensetherapeutics and/or prophylactics. Seemingly diverse organisms such asbacteria, yeast, protozoa, algae, all plant and all higher animal forms,including warm-blooded animals, can be treated by antisense therapy.Further, since each of the cells of multicellular eukaryotes alsoincludes both DNA-RNA transcription and RNA-protein translation as anintegral part of its cellular activity, antisense therapeutics and/ordiagnostics can also be practiced on such cellular populations.Furthermore, many of the organelles, e.g., mitochondria andchloroplasts, of eukaryotic cells also include transcription andtranslation mechanisms. As such, single cells, cellular populations ororganelles can also be included within the definition of organisms thatare capable of being treated with antisense therapeutics or diagnostics.As used herein, therapeutics is meant to include both the eradication ofa disease state, killing of an organism, e.g., bacterial, protozoan orother infection, or control of erratic or harmful cellular growth orexpression.

While we do not wish to be bound by any particular theory, it isbelieved that the presence of many nuclear proteins in the nucleus isdue to their selective entry through the nuclear envelope rather than totheir selective retention within the nucleus after entry. By thismechanism, the nucleus is able to selectively take up certain proteinsand not others. The uptake is based upon the sequence of the peptide orprotein, which provides a selective signal sequence that allowsaccumulation of the peptide or protein in the nucleus. One such peptidesignal sequence is found as part of the SV40 large T-antigen. See, e.g.,Dingwell, et al. Ann. Rev. Cell Bio. 1986, 2, 367; Yoneda, et al.,Experimental Cell Research 1987, 170, 439; and Wychowski, et al., J.Virol. 1986, 61, 3862.

According to the present invention a substituent such as a steroidmolecule, a reporter molecule, a non-aromatic lipophilic molecule, areporter enzyme, a peptide, a protein, a water soluble vitamin, a lipidsoluble vitamin, an RNA cleaving complex, a metal chelator, a porphyrin,an alkylator, a hybrid photonuclease/intercalator, or an aryl azidephoto-crosslinking agent is attached to at least one nucleoside in anantisense diagnostic or therapeutic agent to assist in the transfer ofthe antisense therapeutic or diagnostic agent across cellular membranes.Such antisense diagnostic or therapeutic agent is formed from aplurality of linked nucleosides of a sequence that is “antisense” to aregion of an RNA or DNA that is of interest. Thus, one or morenucleoside of the linked nucleosides are “functionalized” to include asubstituent linked to the nucleoside via a linking group. For thepurposes of identification, such functionalized nucleosides can becharacterized as substituent-bearing (e.g., steroid-bearing)nucleosides. Linked nucleosides having at least one functionalizednucleoside within their sequence demonstrate enhanced antisense activitywhen compared to linked nucleoside that do not contain functionalizednucleoside. These “functionalized” linked nucleosides furtherdemonstrate increased transfer across cellular membranes.

For the purposes of this invention. the terms “reporter molecule” and“reporter enzyme” include molecules or enzymes having physical orchemical properties that allow them to be identified in gels, fluids,whole cellular systems, broken cellular systems, and the like utilizingphysical properties such as spectroscopy, radioactivity, colorimetricassays, fluorescence, and specific binding. Steroids include chemicalcompounds that contain a perhydro-1,2-cyclopentanophenanthrene ringsystem. Proteins and peptides are utilized in their usual sense aspolymers of amino acids. Normally peptides are amino acid polymers thatcontain a fewer amino acid monomers per unit molecule than proteins.Non-aromatic lipophilic molecules include fatty acids, esters, alcoholsand other lipid molecules, as well as synthetic cage structures such asadamantane and buckminsterfullerenes that do not include aromatic ringswithin their structure.

Particularly useful as steroid molecules are the bile acids, includingcholic acid, deoxycholic acid and dehydrocholic acid. Other usefulsteroids are cortisone, digoxigenin, testosterone, cholesterol andcationic steroids such as cortisone having a trimethylaminomethylhydrazide group attached via a double bond at the 3 position of thecortisone rings. Particularly useful reporter molecules are biotin andfluorescein dyes. Particularly useful non-aromatic lipophilic moleculesare alicyclic hydrocarbons, saturated and unsaturated fatty acids,waxes, terpenes, and polyalicyclic hydrocarbons, including adamantaneand buckminsterfullerenes. Particularly useful reporter enzymes arealkaline phosphatase and horseradish peroxidase. Particularly usefulpeptides and proteins are sequence-specific peptides and proteins,including phosphodiesterase, peroxidase, phosphatase, and nucleaseproteins. Such peptides and proteins include SV40 peptide, RNase A,RNase H and Staphylococcal nuclease. Particularly useful terpenoids arevitamin A, retinoic acid, retinal, and dehydroretinol.

Vitamins according to the invention generally can be classified as watersoluble or lipid soluble. Water soluble vitamins include thiamine,riboflavin, nicotinic acid or niacin, the vitamin B6 pyridoxal group,pantothenic acid, biotin, folic acid, the B₁₂ cobamide coenzymes,inositol, choline and ascorbic acid. Lipid soluble vitamins include thevitamin A family, vitamin D, the vitamin E tocopherol family and vitaminK (and phytols). The vitamin A family, including retinoic acid andretinol, are absorbed and transported to target tissues through theirinteraction with specific proteins such as cytosol retinol-bindingprotein type II (CRBP-II), Retinol-binding protein (RBP), and cellularretinol-binding protein (CRBP). These proteins, which have been found invarious parts of the human body, have molecular weights of approximately15 kD. They have specific interactions with compounds of vitamin-Afamily, especially, retinoic acid and retinol.

The vitamin A family of compounds can be attached to oligonucleotidesvia acid or alcohol functionalities found in the various family members.For example, conjugation of an N-hydroxy succinimide ester of an acidmoiety of retinoic acid to an amine function on a linker pendant to anoligonucleotide resulted in linkage of vitamin A compound to theoligonucleotide via an amide bond. Also, retinol was converted to itsphosphoramidite, which is useful for 5′ conjugation.

α-Tocopherol (vitamin E) and the other tocopherols (beta through zeta)can be conjugated to oligonucleotides to enhance uptake because of theirlipophilic character. Also, the lipophilic vitamin, vitamin D, and itsergosterol precursors can be conjugated to oligonucleotides throughtheir hydroxyl groups by first activating the hydroxyls groups to, forexample, hemisuccinate esters. Conjugation then is effected to anaminolinker pendant from the oligonucleotide. Other vitamins that can beconjugated to oligonucleotide aminolinkers through hydroxyl groups onthe vitamins include thiamine, riboflavin, pyridoxine, pyridoxamine,pyridoxal, deoxypyridoxine. Lipid soluble vitamin K's and relatedquinone-containing compounds can be conjugated via carbonyl groups onthe quinone ring. The phytol moiety of vitamin K may also serve toenhance bind of the oligonucleotides to cells.

Pyridoxal (vitamin B₆) has specific B₆-binding proteins. The role ofthese proteins in pyridoxal transport has been studied by Zhang andMcCormick, Proc. Natl. Acad. Sci. USA, 1991 88, 10407. Zhang andMcCormick also have shown that a series of N-(4′-pyridoxyl)amines, inwhich several synthetic amines were conjugated at the 4′-position ofpyridoxal, are able to enter cells by a process facilitated by the B6transporter. They also demonstrated the release of these syntheticamines within the cell. Other pyridoxal family members includepyridoxine, pyridoxamine, pyridoxal phosphate, and pyridoxic acid.Pyridoxic acid, niacin, pantothenic acid, biotin, folic acid andascorbic acid can be conjugated to oligonucleotides usingN-hydroxysuccinimide esters that are reactive with aminolinkers locatedon the oligonucleotide, as described above for retinoic acid.

Other groups for modifying antisense properties include RNA cleavingcomplexes, pyrenes, metal chelators, porphyrins, alkylators, hybridintercalator/ligands and photo-crosslinking agents. RNA cleavers includeo-phenanthroline/Cu complexes and Ru(bipyridine)₃ ²⁺ complexes. TheRu(bpy)₃ ²⁺ complexes interact with nucleic acids and cleave nucleicacids photochemically. Metal chelators are include EDTA, DTPA, ando-phenanthroline. Alkylators include compounds such as iodoacetamide.Porphyrins include porphine, its substituted forms, and metal complexes.Pyrenes include pyrene and other pyrene-based carboxylic acids thatcould be conjugated using the similar protocols.

Hybrid intercalator/ligands include the photonuclease/intercalatorligand6-[[[9-[[6-(4-nitro-benzamido)hexyl]amino]acridin-4-yl]carbonyl]amino]hexanoyl-pentafluorophenylester. This compound has two noteworthy features: an acridine moietythat is an intercalator and a p-nitro benzamido group that is aphotonuclease.

Photo-crosslinking agents include aryl azides such as, for example,N-hydroxysucciniimidyl-4-azidobenzoate (HSAB) andN-succinimidyl-6(-4′-azido-2′-nitrophenyl-amino)hexanoate (SANPAH). Arylazides conjugated to oligonucleotides effect crosslinking with nucleicacids and proteins upon irradiation, They also crosslink with carrierproteins (such as KLH or BSA), raising antibody against theoligonucleotides.

A variety of linking groups can be used to connect the substituents ofthe invention to nucleosides, nucleotides, and/or oligonucleotides.Certain linking groups, such as Ω-aminoalkoxy moieties andΩ-aminoalkylamino moieties, are particularly useful for linking steroidmolecules or reporter molecules to the 2′-position of a nucleoside. Manylinking groups are commercially available, including heterobifunctionaland homobifunctional linking moieties available from the Pierce Co.(Rockford, Ill.). Heterobifunctional and homobifunctional linkingmoieties are particularly useful in conjunction with. the Ω-aminoalkoxyand Ω-aminoalkylamino moieties to form extended linkers that connectpeptides and proteins to nucleosides. Other commercially availablelinking groups are 5′-Amino-Modifier C6 and 3′-Amino-Modifier reagents,both available from Glen Research Corporation (Sterling, Va.).5′-Amino-Modifier C6 is also available from ABI (Applied BiosystemsInc., Foster City, Calif.) as Aminolink-2, and 3′-Amino-Modifier is alsoavailable from Clontech Laboratories Inc. (Palo Alto, Calif.). Anucleotide analog bearing a linking group pre-attached to the nucleosideis commercially available from Glen Research Corporation under thetradename “Amino-Modifier-dT.” This nucleoside-linking group reagent, auridine derivative having an[N(7-trifluoroacetylaminoheptyl)3-acrylamido] substituent group at the 5position of the pyrimidine ring, is synthesized generally according toJablonski, et al., Nucleic Acid Research 1986, 14, 6115. It is intendedthat the nucleoside analogs of the invention include adenine nucleosidesfunctionalized with linkers on their N6 purine amino groups, guaninenucleosides functionalized with linkers at their exocyclic N2 purineamino groups, and cytosine nucleosides functionalized with linkers oneither their N4 pyrimidine amino groups or 5 pyrimidine positions.

Sequence-specific linked nucleosides of the invention are assembled on asuitable DNA synthesizer utilizing either standard nucleotide precursorsor nucleotide precursors that already bear linking moieties. Oncesynthesis of the sequence-specific linked nucleosides is complete, asubstituent can be reacted with the linking moiety. Thus, the inventionpreferably first builds a desired linked nucleoside sequence by knowntechniques on a DNA synthesizer. One or more of the linked nucleosidesare then functionalized or derivatized with a selected substituent.

PCT/US91/00243, application Ser. No. 463,358, and application Ser. No.566,977, which are incorporated herein by reference, disclose thatincorporation of, for example, a 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl,2′-O-allyl, 2′-O-aminoalkyl or 2′-deoxy-2′-fluoro groups on thenucleosides of an oligonucleotide enhance the hybridization propertiesof the oligonucleotide. These applications also disclose thatoligonucleotides containing phosphorothioate backbones have enhancednuclease stability. The functionalized, linked nucleosides of theinvention can be augmented to further include either or both aphosphorothioate backbone or a 2′-O—C₁-C₂₀-alkyl (e.g., 2′-O-methyl,2′-O-ethyl, 2′-O-propyl), 2′-O—C₂-C₂₀-alkenyl (e.g., 2′-O-allyl),2′-O—C₂-C₂₀-alkynyl, 2′-S—C₁-C₂₀-alkyl, 2′-S—C₂-C₂₀-alkenyl,2′-S—C₂-C₂₀-alkynyl, 2′-NH—C₁-C₂₀-alkyl (2′-O-aminoalkyl),2′-NH—C₂-C₂₀-alkenyl, 2′-NH—C₂-C₂₀-alkynyl or 2′-deoxy-2′-fluoro group.See, e.g., application Ser. No. 918,362, filed Jul. 23, 1992, which isincorporated by reference.

An oligonucleotide possessing an amino group at its 5′-terminus isprepared using a DNA synthesizer and then is reacted with an activeester derivative of the substituent of the invention (e.g., cholicacid). Active ester derivatives are well known to those skilled in theart. Representative active esters include N-hydrosuccinimide esters,tetrafluorophenolic esters, pentafluorophenolic esters andpentachlorophenolic esters. For cholic acid, the reaction of the aminogroup and the active ester produces an oligonucleotide in which cholicacid is attached to the 5′-position through a linking group. The aminogroup at the 5′-terminus can be prepared conveniently utilizing theabove-noted 5′-Amino-Modifier C6 reagent.

Cholic acid can be attached to a 3′-terminal amino group by reacting a3′-amino modified controlled pore glass (sold by Clontech LaboratoriesInc., Palo Alto, Calif.), with a cholic acid active ester.

Cholic acid can be attached to both ends of a linked nucleoside sequenceby reacting a 3′,5′-diamino sequence with the cholic acid active ester.The required oligonucleoside sequence is synthesized utilizing the3′-Amino-Modifier and the 5′-Amino-Modifier C6 (or Aminolink-2) reagentsnoted above or by utilizing the above-noted 3′-amino modified controlledpore glass reagent in combination with the 5′-Amino-Modifier C2 (orAminolink-2) reagents.

In even further embodiments of the invention, an oligonucleosidesequence bearing an aminolinker at the 2′-position of one or moreselected nucleosides is prepared using a suitably functionalizednucleotide such as, for example,5′-dimethoxytrityl-2′-O-(ε-phthalimidylamino-pentyl)-2′-deoxyadenosine-3′-N,N-diisopropyl-cyanoethoxyphosphoramidite. See, e.g., Manoharan, et al., Tetrahedron Letters,1991, 34, 7171 and above-referenced Application Serial Nos.PCT/US91/00243, 566,977, and 463,358. The nucleotide or nucleotides areattached to cholic acid or another substituent using an active ester ora thioiso-cyanate thereof. This approach allows the introduction of alarge number of functional groups into an linked nucleoside sequence.Indeed each of the nucleosides can be so substituted.

In further functionalized nucleoside sequences of the invention, theheterocyclic base of one or more nucleosides is linked to a steroidmolecule, a reporter molecule, a non-aromatic lipophilic molecule, areporter enzyme, a peptide, a protein, a water soluble vitamin, a lipidsoluble vitamin, an RNA cleaving complex, a metal chelator, a porphyrin,an alkylator, a hybrid photonuclease/intercalator, or an aryl azidephoto-crosslinking agent. Utilizing5′-O-dimethoxytrityl-5-[N(7-trifluoroacetylaminoheptyl)-3-acryl-amido]-2′-deoxyuridine3′-O-(methyl N,N-diisopropyl)phosphoramide, as described by Jablonski,et al. above (also commercially available from Glen Research), thedesired nucleoside, functionalized to incorporate a linking group on itsheterocyclic base, is incorporated into the linked nucleoside sequenceusing a DNA synthesizer.

Conjugation (linking) of reporter enzymes, peptides, and proteins tolinked nucleosides is achieved by conjugation of the enzyme, peptide orprotein to the above-described amino linking group on the nucleoside.This can be effected in several ways. A peptide- orprotein-functionalized nucleoside of the invention can be prepared byconjugation of the peptide or protein to the nucleoside usingEDC/sulfo-NHS (i.e.,1-ethyl-3(3-dimethylaminopropylcarbodiimide/N-hydroxysulfosuccinimide)to conjugate the carboxyl end of the reporter enzyme, peptide, orprotein with the amino function of the linking group on the -nucleotide.Further, a linked nucleoside sequence of the invention can be preparedusing EDC/sulfo-NHS to conjugate a carboxyl group of an aspartic orglutamic acid residue in the reporter enzyme, peptide or protein to theamino function of a linked nucleoside sequence.

Preferably a reporter enzyme-, peptide-, protein-functionalized linkednucleoside sequence can be prepared by conjugation of the reporterenzyme, peptide or protein to the nucleoside sequence via aheterobifunctional linker such asm-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (MBS) or succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) to link a thiolfunction on the reporter enzyme, peptide or protein to the aminofunction of the linking group on nucleoside sequence. By this mechanism,an oligonucleoside-maleimide conjugate is formed by reaction of theamino group of the linker on the linked nucleosides with the MBS or SMCCmaleimide linker. The conjugate is then reacted with peptides orproteins having free sulfhydryl groups.

In a second preferred method, a reporter enzyme-, peptide-,protein-functionalized linked nucleoside sequence can be prepared byconjugation of the peptide or protein to the sequence using ahomobifunctional linker such as disuccinimidyl suberate (DSS) to link anamino function on the peptide or protein to the amino group of a linkeron the sequence. By this mechanism, an oligonucleoside-succinimidylconjugate is formed by reaction of the amino group of the linker on thenucleoside sequence with a disuccinimidyl suberate linker. Thedisuccinimidyl suberate linker couples with the amine linker on thesequence to extend the size of the linker. The extended linker is thenreacted with amine groups such as, for example., the amine of lysine orother available N-terminus amines, on reporter enzymes, peptides andproteins.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples, which are not intended to be limiting.

For the following examples, anhydrous dimethylformamide, cholic acid andN-hydroxysuccinimide were purchased from Aldrich Chemical Co.(Milwaukee, Wis.), ethyl-3-(3-dimethylamino)propylcarbodiimide (EDAC orEDC) was obtained from JBL Scientific (San Luis Obispo, Calif.) as thefree base under the label EDAC or from Pierce (Rockford, Ill.) under thelabel EDC, Aminolink-2 was purchased from ABI and 3′-Amino-Modifier,5′-Amino-Modifier C6 and Amino-Modifier dT reagents were purchased fromGlen Research Corporation. NMR Spectra were run on a Varian Unity-400instrument. Oligonucleotide synthesis were-performed on an AppliedBiosystems 380 B or 394 DNA synthesizer following standardphosphoramidite protocols using reagents supplied by the manufacturer.When modified phophoramidites were used, a longer coupling time (10-15min) was employed. HPLC was performed on a Waters 600E instrumentequipped with a model 991 detector. Unless otherwise noted, foranalytical chromatography the following conditions were employed:Hamilton PRP-1 column (15×2.5 cm); solvent A: 50 mm TEAA, pH 7.0;solvent B: 45 mm TEAA with 80% CH₃CN; flow rate: 1.5 ml/min; gradient:5% B for the first 5 minutes, linear (1%) increase in B every minutethereafter and for preparative purposes: Waters Delta Pak C-4 column;flow rate: 5 ml/min; gradient: 5% B for the first 10 minutes, linear 1%increase for every minute thereafter.

All oligonucleotide sequences are listed in a standard 5′ to 3′ orderfrom left to right.

EXAMPLE 1 Cholic Acid N-Hydroxysuccinimide Ester (Compound 1)

Anhydrous DMF (150 ml) was added to a mixture of cholic acid (4.09 g, 15mmol) and N-hydroxysuccinimide (5.25 g, 45 mmol). The mixture wasstirred in the presence of nitrogen. EDAC (4 ml, 25 mmol) was then addedand this mixture was then stirred overnight. The solution was thenevaporated to a gum and partitioned between 1:1 ethyl acetate and 4%NaHCO₃ solution (pH 7.9) (100 ml each). The organic layer was washedwith saturated NaCl solution, dried over anhydrous Na₂SO₄ and evaporatedto yield the title compound as a pale yellow foam (4.6 g, 91%). ¹³C NMR(DMSO-d₆) δ 12.27, 16.71, 22.58, 22.80, 25.42, 26.19, 27.20, 28.49,30.41, 30.56, 34.36, 34.82, 34.82, 35.31, 39.09, 39.09, 41.38, 41.53,45.84, 46.05, 66.25, 70.45, 71.03, 169.28 and 170.16.

EXAMPLE 2 Heterocyclic Bas Cholic Acid End-Labeled Oligodeoxynucleotide

An oligonucleotide of the sequence: Oligomer 1: TTG CTT* CCA TCT TCC TCGTCwherein T* represents a nucleotide functionalized to included a cholicacid linked via a linker to the heterocyclic base of a 2′-deoxyuridinenucleotide was prepared in a 1 μmol scale. Oligomer 1 is useful as anHPV antisense oligonucleotide.

A. Preparation of Intermediate Linker

The linker of the structure —CH═CH—C(O)—NH—(CH₂)₆—NH—C(O)—CF₃ wasintroduced via a suitable protected and activated 2′-deoxyuridinephosphoramide intermediate obtained from Glen Research Corporation asAmino-Modifier-dT. The oligonucleotide bearing the linker thereon wasdeprotected and purified by HPLC.

B. Preparation of Cholic Acid Functionalized Oligonucleotide

An aliquot of the linker bearing oligonucleotide of Example 2-A(approximately 100 O.D. units, 550 nmols) was dissolved in 500 μl of0.2M NaHCO₃ buffer and to this solution cholic acid N-hydroxysuccinimideester (Compound 1, 75 mg, 149 μmols) was added and heated at 45° C.overnight. It was then passed through a Sephadex G-25 (1.0×25 cm)column. Concentration of the oligonucleotide fractions to 2 ml followedby HPLC purification yielded the desired conjugate wherein cholic acidis internally linked at C-5 of the heterocyclic base.

EXAMPLE 3 5′-Terminus Cholic Acid End-Labeled Oligodeoxynucleotide

A phosphorothioate oligonucleotide having cholic acid attached to its5′-terminus of the oligonucleotide sequence:

Oligomer 2: 5′-CHA-C_(s)T_(s)G_(s) T_(s)C_(s)T_(s) C_(s)C_(s)A_(s)T_(s)C_(s)T_(s) T_(s)C_(s)A_(s) C_(s)Twherein CHA represents cholic acid and the subscript “s” represents aphosphorothioate inter-nucleotide backbone linkage was prepared.

A. Preparation of Intermediate Linker

The oligonucleotide sequence having a 5′-terminus amino group wassynthesized on a 3×1.0 μmol scale in the standard manner on the DNAsynthesizer utilizing phosphoramidite methodology. The phosphorothioateintra-nucleotide backbone was formed using a phosphorothioate reagent(Beaucage reagent, i.e., 3H-1,2-benzodithioate-3-one 1,1-dioxide; see,Radhakrishnan, et al., J. Am. Chem. Soc. 1990, 112, 1253). TheAminolink-2 reagent was used at the last step of the oligonucleotidesynthesis. Deprotection with concentrated NH₄OH for 16 hrs at 55° C.yielded the 5′-aminolinker-oligonucleotide.

B. Preparation of Cholic Acid Functionalized Oligonucleotide

The crude 5′-aminolinker-oligonucleotide of Example 3-A (100 O.D. units,approximately 600 nmols based on the calculated extinction coefficientof 1.6756×10⁵ at 260 nm) was dissolved in freshly prepared NaHCO₃ buffer(500 μl, 0.2M, pH 8.1) and treated. with a solution of cholic acidN-hydroxysuccinimide ester (Compound 1, 75 mg, 149 μmols) dissolved in200 μl of DMF. The reaction mixture was heated at 45° C. overnight. Itwas then passed through a Sephadex G-25 (1.0×25 cm) column.Concentration of the oligonucleotide fractions to 2 ml followed by HPLCpurification yielded 54 OD units of the desired conjugate (54% yield).HPLC retention times were: 37.42 for the unreacted oligonucleotide andany failure sequences produced during oligonucleotide synthesis and54.20 for the final product.

EXAMPLE 4 3′-Terminus Cholic Acid End Labeled Oligodeoxynucleotide

A. 3′-Terminus Cholic Acid Oligonucleotide

A phosphorothioate oligonucleotide having cholic acid attached to its3′-terminus of the oligonucleotide sequence: Oligomer 3: C_(s)T_(s)G_(s)T_(s)C_(s)T_(s) C_(s)C_(s)A_(s) T_(s)C_(s)C_(s) T_(s)C_(s)T_(s)T_(s)C_(s)A C_(s)T 3′-CHAwherein CHA represents cholic acid and the subscript “s” represents aphosphorothioate inter-nucleotide backbone linkage was prepared.

1. Preparation of Intermediate Linker

The oligonucleotide sequence having a 3′-terminus amino group wassynthesized using 3′-amino modifier controlled pore glass (CPG) fromClontech Laboratories (Palo Alto, Calif.) as the solid support. Theabove-noted Beaucage reagent was utilized to form the phosphorothioateinter-nucleotide backbone. The synthesis was conducted in a “Trityl-Off”mode. The resultant solid support was deprotected with concentratedNH₄OH for 16 hrs at 55° C. Purification on a Sephadex G-25 columnyielded a 3′-amino functionalized phosphorothioate oligonucleotide ofthe specified oligonucleotide sequence.

2. Preparation of Cholic Acid Functionalized Oligonucleotide

The crude oligonucleotide of Example 4-A-1 (50 O.D. units, approximately300 nmols) was reacted with cholic acid N-hydroxysuccinimide ester(Compound 1, 40 mg) as per the procedure of Example 3. HPLC retentiontimes were 37.45 for the starting oligonucleotide material and 51.68 forthe product.

B. 3′-Terminus Cholic Acid Oligonucleotide

A phosphorothioate oligonucleotide having cholic acid attached to its3′-terminus and of the oligonucleotide sequence: Oligomer 4:T_(s)G_(s)G_(s) G_(s)A_(s)G_(s) C_(s)C_(s)A_(s) T_(s)A_(s)G_(s)C_(s)G_(s)A_(s) G_(s)G_(s)C 3′-CHAwherein CHA represents cholic acid and the subscript “s” represents aphosphorothioate inter-nucleotide backbone linkage was prepared in thesame manner as for the oligonucleotide of Example 4-A-2.

EXAMPLE 5 3′-Terminus Cholic Acid & 5′-Terminus Cholic AcidDi-End-Labeled Oligodeoxynucleotide

A phosphorothioate oligonucleotide having cholic acid attached to bothof the 3′-terminus and the 5′-terminus of the oligonucleotide sequence:Oligomer 5: 5′-CHA C_(s)T_(s)G_(s) T_(s)C_(s)T_(s) C_(s)C_(s)A_(s)T_(s)C_(s)T_(s) T_(s)C_(s)A_(s) C_(s)T 3′-CHAwherein CHA represents cholic acid and the subscript “s” represents aphosphorothioate inter-nucleotide backbone linkage was synthesized on a3×1.0 μmol scale. Oligomer 5 has the same sequence as Oligomer 1 exceptfor the cholic acid functionalization.

A. Preparation of Intermediate Linker

The oligonucleotide synthesis was conducted using a 3′-amino modifiedcontrolled pore glass (CPG) from Clontech Laboratories as the solidsupport. Oligonucleotide synthesis was conducted utilizingphosphoramidite synthetic methodology and the Beaucage reagent to form aphosphorothioate inter-nucleotide backbone as per Example 4 above. Uponcompletion of the basic sequence and while still on the DNA synthesizer,Aminolink-2 reagent was used to introduce a 5′-terminus aminofunctionality on to the oligonucleotide. Deprotection with concentratedammonium hydroxide and purification on a Sephadex G-25 column yielded3′,5′-diaminolinker oligonucleotide.

B. Preparation of Cholic Acid Functionalized Oligonucleotide

The crude di-aminolinker oligonucleotide (50 O.D. units, approximately300 nmols, based on the calculated extinction coefficient of 1.6756×10⁵at 260 nm) was dissolved in freshly prepared NaHCO₃ buffer (500 μl,0.2M, pH 8.1) and treated with a solution of cholic acidN-hydroxysuccinimide ester (Compound 1, 50 mg, 98.6 μmols) dissolved in200 μl of DMF. The reaction mixture was heated at 45° C. overnight. Itwas then passed through a Sephadex G-25 (1.0×25 cm) column. Theoligonucleotide fractions were concentrated to 2 ml and purified byreverse phase HPLC. Retention times were 37.76 for unreactedoligonucleotide, 51.65 for 3′-cholic acid conjugated oligonucleotide,54.34 for 5′-cholic acid conjugated oligonucleotide and 58.75 for3′,5′-di-cholic acid conjugated oligonucleotide. The 58.75 min. productwas desalted on a Sephadex G-25 column to yield 11 O.D units (22%) ofthe desired product.

EXAMPLE 6 3′-Terminus Cholic Acid or 5′-Terminus Cholic AcidFunctionalized, 2′-O-Methyl Derivatized Oligodeoxynucleotides

Phosphorothioate oligonucleotides having cholic acid attached to eitherthe 3′-terminus end or the 5′-terminus end of the oligonucleotidesequence and further being uniformly functionalized to include a2′-O-methyl group on each of the nucleotides of the oligonucleotide weresynthesized. The following oligonucleotides having uniform 2′-O-methylsubstitutions were synthesized: Oligomer 6: 5′-CHA CCC AGG CUC AGA 3′;Oligomer 7: 5′ CCC AGG CUC AGA 3′-CHA; and Oligomer 8: 5′-CHA GAG CUCCCA GGC 3′.

A. Preparation of Intermediate Linker

Synthesis of the intermediate 5′ or 3′-aminolinker oligonucleotide wasconducted utilizing 2′-O-methyl phosphoramidite nucleotides availablefrom Chemgenes Inc. (Needham, Mass.) and phosphoramidite chemistry asper Examples 3 and 4 above, respectively. Each of the intermediateoligonucleotides were deprotected in concentrated NH₄OH, evaporated andde-salted on a Sephadex G-25 column.

B. Preparation of Cholic Acid Functionalized Oligonucleotide

The resultant crude oligonucleotides from Example 6-A-1 (from 30 to 40O.D. units, 250-350 nmols based on calculated extinction coefficients of1.1797×10⁵, 1.1777×10⁵ and 1.1481×10⁵ at 260 nm, respectively forOligomers 6, 7 and 8) were dried and dissolved in 250 μl of 0.2M NaHCO₃buffer and treated with a solution of cholic acid N-hydroxysuccinimideester (Compound 1, from 30 to 40 mg, 60 to 80 μmols) dissolved in 500 μlof 0.2 M NaHCO₃ buffer and 200 μl DMF and heated between 40-45° C. for12-24 hrs. The reaction mixtures were evaporated and dissolved in 2 mlof water and washed with 3×2 ml of ethyl acetate. The resultant aqueoussolutions were purified by reverse phase HPLC. For Oligomer 6 the HPLCretention times were 34.65 for the starting oligonucleotide material and58.75 for the product; for oligomer 7 the HPLC retention times were37.23 for the starting oligonucleotide material and 55.32 for theproduct; and for Oligomer 8 the HPLC retention times were 34.99 for thestarting oligonucleotide material and 56.98 for the product. Theproducts were evaporated and desalted on a Sephadex G-25 column. Theyield averaged about 20% in each case.

EXAMPLE 7 Oligonucleotides Having 2′-Protected-Amine Terminating LinkingGroup

A. Preparation of5′-Dimethoxytrityl-2′-(O-Pentyl-N-phthalimido)-2′-DeoxyadenosinePhosphoramidite (Compound 2)

To introduce a functionalization at the 2′ position of nucleotideswithin desired oligonucleotide sequences,5′-Dimethoxytrityl-2′-(O-pentyl-N-phthalimido)-2′-deoxyadenosinephosphoramidite (Compound 2) was utilized to provide a linking groupattached to the 2′ position of nucleotide components of anoligonucleotide. Compound 2 was synthesized as per the procedures ofpatent applications US91/00243 and 463,358, identified above startingfrom adenosine. Briefly this procedure treats adenosine with NaH in DMFfollowed by treatment with N-(5-bromopentyl)phthalimide. Furthertreatment with (CH₃)₃SiCl, Ph-C(O)—Cl and NH₄OH yields N6-benzylprotected 2′-pentyl-N-phthalimido functionalized adenosine. Treatmentwith DIPA and CH₂Cl₂ adds a DMT blocking group at the 5′ position.Finally phosphitylation gives the desired phosphoramidite compound,Compound 2. Compound 2 was utilized in the DNA synthesizer as a 0.09Msolution in anhydrous CH₃CN. Oligonucleotide synthesis was carried outin either an ABI 390B or 394 synthesizer employing the standardsynthesis cycles with an extended coupling time of 10 minutes duringcoupling of Compound 2 into the oligonucleotide sequence. Couplingefficiency of >98% was observed for Compound 2 coupling.

B. 2′-Protected-Amine Linking Group Containing PhosphodiesterOligonucleotides

The following oligonucleotides having phosphodiester inter-nucleotidelinkages were synthesized: Oligomer 9: 5′ TA*G 3′; Oligomer 10: 5′ CCA*G 3′; Oligomer 11: 5′ GGC TGA* CTG CG 3′; Oligomer 12: CTG TCT CCA* TCCTCT TCA CT; and Oligomer 13: CTG TCT CCA* TCC TCT TCA* CTwherein A* represents a nucleotide functionalized to incorporate apentyl-N-phthalimido functionality. Oligomers 12 and 13 are antisensecompounds to the E2 region of the bovine papilloma virus-1 (BPV-1).Oligomers 12 and 13 have the same sequence as Oligomer 3 except for the2′ modification. The oligonucleotides were synthesized in either a 10μmol scale or a 3×1 μmol scale in the “Trityl-On” mode. Standarddeprotection conditions (30% NH₄OH, 55° C., 24 hr) were employed. Theoligonucleotides were purified by reverse phase HPLC (Waters Delta-PakC₄ 15 μm, 300 A, 25×100 mm column equipped with a guard column of thesame material). They were detritylated and further purified by sizeexclusion using a Sephadex G-25 column. NMR analyses by both proton andphosphorus NMR confirmed the expected structure for the Oligomers 9 and10.

C. 2′-Protected-Amine Linking Group Containing PhosphorothioateOligonucleotides

The following oligonucleotides having phosphorothioate inter-nucleotidelinkages were synthesized: Oligomer 14: T_(s)T_(s)G_(s) C_(s)T_(s)T_(s)C_(s)C_(s)A*_(s) T_(s)C_(s)T_(s) T_(s)C_(s)C_(s) T_(s)C_(s)G_(s) T_(s)C;Oligomer 15: T_(s)G_(s)G_(s) G_(s)A_(s)G_(s) C_(s)C_(s)A_(s)T_(s)A_(s)G_(s) C_(s)G_(s)A*_(s) G_(s)G_(s)C; and Oligomer 16:T_(s)G_(s)G_(s) G_(s)A*_(s)G_(s) C_(s)C_(s)A*_(s) T_(s)A*_(s)G_(s)C_(s)G_(s)A*_(s) G_(s)G_(s)Cwherein A* represents a nucleotide functionalized to incorporate apentyl-N-phthalimido functionality and the subscript “is” represents aphosphorothioate inter-nucleotide backbone linkage. Oligomer 14 is anantisense compound directed to the E2 region of the bovine papillomavirus-1 (BPV-1). Oligomers 15 and 16 are antisense compounds to ICAM.Oligomer 14 has the same sequence as Oligomer 3 except for the 2′modification whereas Oligomers 15 and 16 have the same sequence asOligomer 4 except for the 2′ modification. These oligonucleotides weresynthesized as per the method of Example 7-B except during thesynthesis, for oxidation of the phosphite moieties, the Beaucage reagent(see Example 3 above) was used as a 0.24 M solution in anhydrous CH₃CNsolvent. The oligonucleotides were synthesized in the “Trityl-On” modeand purified by reverse phase HPLC utilizing the purification procedureof Example 7-B.

D. 2′-O-Methyl Derivatized, 2′-Protected-Amine Linking Group ContainingRNA Oligonucleotides.

The following oligonucleotides having 2′-O-methyl groups on eachnucleotide not functionalized with a 2′-protected aminefunctionalization were synthesized: Oligomer 17: CCA A*GC CUC AGA; andOligomer 18: CCA GGC UCA GA*Twherein A* represents a nucleotide functionalized to incorporate apentyl-N-phthalimido functionality and where the remaining nucleotidesexcept the 3′-terminus nucleotide are each 2′-O-methyl derivatizednucleotides. The 3′-terminus nucleotide in both Oligomers 17 and 18 is a2′-deoxy nucleotide. Both Oligomers 17 and 18 are antisense compounds tothe HIV-1 TAR region. The oligonucleotides were synthesized as per themethod of Example 6 utilizing Compound 2 for introduction of thenucleotides containing the pentyl-N-phthalimido functionality andappropriate 2-O-methyl phosphoramidite nucleotides from Chemgenes Inc.(Needham, Mass.) for the remaining RNA nucleotides. The 3′-terminusterminal 2′-deoxy nucleotides were standard phosphoamidites utilized forthe DNA synthesizer. The oligonucleotides were deprotected and purifiedas per the method of Example 7-B.

EXAMPLE 8 Functionalization of Oligonucleotides at the 2′ Position

A. Functionalization with Biotin

1. Single Site Modification

About 10 O.D. units (A₂₆₀) of oligomer 12 (see Example 7) (approximately60 nmols based on the calculated extinction coefficient of 1.6756×10⁵)was dried in a microfuge tube. The oligonucleotide was dissolved in 200μl of 0.2 M NaHCO₃ buffer and D-biotin-N-hydroxysuccinimide ester (2.5mg, 7.3 μmols) (Sigma, St. Louis, Mo.) was added followed by 40 μl DMF.The solution was let stand overnight. The solution was applied to aSephadex G-25 column (0.7×15 cm) and the oligonucleotide fractions werecombined. Analytical HPLC showed nearly 85% conversion to the product.The product was purified by HPLC (Waters 600E with 991 detector,Hamilton PRP-1 column 0.7×15 cm; solvent A: 50 mM TEAA pH 7.0; B: 45 mMTEAA with 80% acetonitrile: 1.5 ml flow rate: Gradient: 5% B for first 5mins., linear (1%) increase in B every minute thereafter) and furtherdesalted on Sephadex G-25 to give the oligonucleotide: Oligomer 19: CTGTCT CCA* TCC TCT TCA CTwherein A* represents a nucleotide functionalized to incorporate abiotin functionality linked via a 2′-O-pentyl-amino linking group to the2′ position of the designated nucleotide. HPLC retention times are shownin Table 1 below.

2. Multiple Site Modification

About 10 O.D. units (A₂₆₀) of Oligomer 13 (see Example 7, approximately60 nmols) was treated utilizing the method of Example 8-A-1 withD-biotin-N-hydroxysuccinimide ester (5 mg) in 300 μl of 0.2 M NaHCO₃buffer/50 μl DMF. Analytical HPLC showed 65% of double labeledoligonucleotide product and 30% of single labeled products (from the twoavailable reactive sites). HPLC and Sephadex G-25 purification gave theoligonucleotide: Oligomer 20: CTG TCT CCA* TCC TCT TCA* CTwherein A* represents nucleotides functionalized to incorporate a biotinfunctionality linked via a 2′-O-pentyl-amino linking group to the 2′position of the designated nucleotide. HPLC retention times for thisproduct (and its accompanying singly labeled products) are shown inTable 1 below.

B. Functionalization with Fluorescein

1. Single Site Modification

A 1M Na₂CO_(3/1)M NaHCO₃ buffer (pH 9.0) was prepared by adding 1MNaHCO₃ to 1 M Na₂CO₃. 200 μl of this buffer was added to 10 O.D. unitsof Oligomer 12 (see Example 7) in a microfuge tube. 10 mg offluorescein-isocyanate in 500 μl DMF was added to give a 0.05 Msolution. 100 μl of the fluorescein solution was added to theoligonucleotide solution in the microfuge tube. The tube was coveredwith aluminum foil and let stand overnight. The reaction mixture wasapplied to a Sephadex G-25 column (0.7×20 cm) that had been equilibratedwith 25% (v/v) ethyl alcohol in water. The column was eluted with thesame solvent. Product migration could be seen as a yellow band wellseparated from dark yellow band of the excess fluorescein reagent. Thefractions showing absorption at 260 nm and 485 nm were combined andpurified by HPLC as per the purification procedure of Example 8-A-1.Analytical HPLC indicated 81% of the desired doubly functionalizedoligonucleotide. The product was lyophilized and desalted on Sephadex togive the oligonucleotide: Oligomer 21: CTG TCT CCA* TCC TCT TCA CTwherein A* represents a nucleotide functionalized to incorporate afluorescein functionality linked via a 2′-O-pentyl-amino linking groupto the 2′ position of the designated nucleotide. HPLC retention timesare shown in Table 1 below.

2. Multiple Site Modification

10 O.D. units (A₂₆₀) of Oligomer 13 (from Example 7) was dissolved in300 μl of the 1M Na₂HCO_(3/1)M Na₂CO₂ buffer of Example 8-B-1 and 200 μlof the fluorescein-isothiocyanate stock solution of Example 8-B-1 wasadded. The resulting solution was treated as per Example 8-B-1.Analytical HPLC indicated 61% of doubly labeled product and 38% ofsingly labeled products. Work up of the reaction gave theoligonucleotide: Oligomer 22: CTG TCT CCA* TCC TCT TCA* CTwherein A* represents nucleotides functionalized to incorporate afluorescein functionality linked via a 2′-O-pentyl-amino linking groupto the 2′ position of the designated nucleotide. HPLC retention timesare shown in Table 1 below.

C. Functionalization with Cholic Acid

1. Single Site Modification

10 O.D. units (A₂₆₀) of Oligomer 12 (see Example 7) was treated withcholic acid-NHS ester (Compound 1, 5 mg, 9.9 μmols) in 200 μl of 0.2 MNaHCO₃ buffer/40 μl DMF. The reaction mixture was heated for 16 hrs at45° C. The product was isolated as per the method of Example 8-A-1.Analytical HPLC indicated >85% product formation. Work up of thereaction gave the oligonucleotide: Oligomer 23: CTG TCT CCA* TCC TCT TCACTwherein A* represents a nucleotide functionalized to incorporate acholic acid functionality linked via a 2′-O-pentyl-amino linking groupto the 2′ position of the designated nucleotide. HPLC retention timesare shown in Table 1 below.

2. Multiple Site Modification

10 O.D. units (A₂₆₀) of Oligomer 13 (see Example 7) was treated withcholic acid-NHS ester (Compound 1, 10 mg, 19.8 μmols) in 300 μl of 0.2 MNaHCO₃ buffer/50 μl DMF. The reaction mixture was heated for 16 hrs at45° C. The product was isolated as per the method of Example 8-A-1.Analytical HPLC revealed 58% doubly labeled product, 17% of a firstsingly labeled product and 24% of a second singly labeled product. Workup as per Example 8-A-1 gave the oligonucleotide: Oligomer 24: CTG TCTCCA* TCC TCT TCA* CTwherein A* represents nucleotides functionalized to incorporate a cholicacid functionality linked via a 2′-O-pentyl-amino linking group to the2′ position of the designated nucleotide. HPLC retention times are shownin Table 1 below.

D. Functionalization with Digoxigenin

1. Single Site Modification

10 O.D. units (A₂₆₀) of Oligomer 12 (see Example 7) was treated withdigoxigenin-3-O-methylcarbonyl-ε-aminocaproic N-hydroxy succinimideester (Boehringer Mannheim Corporation, Indianapolis, Ind.) in 200 μl of0.1 M borate pH 8.3 buffer/40 μl DMF. The reaction mixture was let standovernight. The product was isolated as per the method of Example 8-A-1.Work up of the reaction gave the oligonucleotide: Oligomer 25: CTG TCTCCA* TCC TCT TCA CTwherein A* represents a nucleotide functionalized to incorporate adigoxigenin functionality linked via a 2′-O-pentyl-amino linking groupto the 2′ position of the designated nucleotide. HPLC retention timesare shown in Table 1 below.

2. Multiple Site Modification

10 O.D. units (A₂₆₀) of Oligomer 13 (see Example 7) was treated withdigoxigenin-3-O-methylcarbonyl-ε-aminocaproic N-hydroxy succinimideester (Boehringer Mannheim Corporation, Indianapolis, Ind.) in 300 μl of0.1 M borate pH 8.3 buffer/50 μl DMF. The reaction mixture was let standovernight. The product was isolated as per the method of Example 8-A-1.Work up as per Example 8-A-1 gave the oligonucleotide: Oligomer 26: CTGTCT CCA* TCC TCT TCA* CT

wherein A* represents nucleotides functionalized to incorporate a cholicacid functionality linked via a 2′-O-pentyl-amino linking group to the2′ position of the designated nucleotide. HPLC retention times are shownin Table 1 below. TABLE 1 HPLC Retention Times Of OligonucleotidesFunctionalized At 2′ Position Retention Time Minutes Oligomer MonoSubstitution Multiple Substitution Oligomer 12¹ 21.78 Oligomer 13¹ 22.50Oligomer 19² 23.58 Oligomer 20² 24.16^(a) 25.19^(b) Oligomer 21³ 26.65Oligomer 22³ 26.99^(a) 29.33^(b) 27.55^(a) Oligomer 23⁴ 30.10 Oligomer24⁴ 30.38^(a) 37.00^(b) 32.22^(a) Oligomer 25⁵ 28.06 Oligomer 26⁵28.14^(a) 33.32^(b) 29.24^(a)Conditions: Waters 600E with 991 detector, Hamilton PRP-1 column 0.7 ×15 cm; solvent A: 50 mM TEAA pH 7.0; B: 45 mM TEAA with 80%acetonitrile: 1.5 ml flow rate: Gradient: 5% B for first 5 mins., linear(1%) increase in B every minute thereafter;^(a)Mono conjugated minor product;^(b)Doubly conjugated major product;¹Parent Oligonucleotide - no 2′ functionalization;²2′ Biotin functionalization;³2′ Fluorescein functionalization;⁴2′ Cholic Acid functionalization; and⁵2′ Digoxigenin functionalization.

EXAMPLE 9 Functionalization of Oligonucleotide at the 2′ Position withReporter Enzymes, Peptides and Proteins

A. Use of Heterobifunctional Linker

1. Synthesis of Oligonucleotide-Maleimide Conjugate

Oligomer 12 (Example 7) (100 O.D. units, 600 nmols) is lyophilized in a5 ml pear-shaped flask. Sulfo-SMCC reagent, Pierce Chemical Co.(Rockford, Ill.) (16 mg, 46 μmols) is dissolved in phosphate buffer (800μl, 0.1M, pH 7.0) and added to the oligonucleotide bearing flask. Anadditional 200 μl of buffer are used to wash the reagent and transfer itto the oligonucleotide flask. The contents of the flask are stirredovernight and loaded on to a Sephadex G-25 column (1×40 cm) equippedwith a fraction collector. The oligonucleotide-maleimide conjugatecontaining fractions are collected and tested by analytical HPLC forseparation from other NHS type products.

2. Synthesis of Oligonucleotide-Peptide Conjugate

An aliquot of the oligonucleotide-maleimide conjugate of Example 9-A-1(about 50 O.D. units, 300 nmols) is lyophilized in a microfuge tube.SV40 peptide (pro-asp-lys-lys-arg-lys-cys) (2.5 mg, about 2.5 μmols) istaken up in phosphate buffer (800 μl, 0.1 M, pH 7.0) and added to theoligonucleotide-maleimide conjugate containing tube. The contents of thetube are stirred overnight under an argon atmosphere. The reactionmixture is passed through a Sephadex G-25 column and theoligonucleotide-peptide conjugate fractions are identified by HPLC.Isolation of the product from product-bearing fractions via HPLC anddesalting on Sephadex G-25 will yield an oligonucleotide of thesequence: Oligomer 27: CTG TCT CCA* TCC TCT TCA CTwherein A* represents a nucleotide functionalized to incorporate a SV40peptide functionality linked via a 2′-O-pentyl-amino-sulfo-SMCC(sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate)linking group to the 2′ position of the designated nucleotide.

B. Use of Homobifunctional Linker

1. Synthesis of Oligonucleotide-Disuccinimidyl Suberate Conjugate

An aliquot (10 O.D. units, 60 nmols) of Oligomer 12 (Example 7) isevaporated to dryness and is dissolved in freshly prepared 0.1 MNaHCO₃/50 mM EDTA (100 μl, pH 8.25). The solution is then treated with asolution of DSS, Pierce Chemical Co. (Rockford, Ill.) (2.6 mg, 7 μmol)in 200 μl DMSO. The solution is stored at room temperature for 15minutes and then immediately applied to a Sephadex G-25 column (1×40 cm)that is previously packed and washed with water at 4° C. Theoligonucleotide fractions are combined immediately in a 25 mlpear-shaped flask and are rapidly frozen in dry ice/isopropyl alcoholand lyophilized to a powder.

2. Synthesis of Oligonucleotide-Protein Conjugate

A solution of calf intestinal alkaline phosphatase (Boehringer Mannheim)(20.6 mg, 2.06 ml, 147 nmol) is spun at 4° C. in a Centriconmicroconcentrator at 6000 rpm until the volume is less than 50 μl. It isthen redissolved in 1 ml of cold Tris buffer (pH 8.5, O.1M containing0.1 NaCl and 0.05 M MgCl₂) and concentrated twice more. Finally theconcentrate is dissolved in 400 μl of the same buffer. This solution isadded to the activated oligonucleotide from Example 9-B-1 and thesolution stored for 18 hrs at room temp. The product is diluted toapproximately 30 ml and applied to a Sephadex G-25 column (1×20 cm,chloride form) maintained at 4° C. The column is eluted with 50 nMTris-Cl pH 8.5 until the UV absorbance of the fractions eluted reachnear zero values. The column is then eluted with a NaCl salt gradient0.05 M to 0.75 M. (150 ml each). The different peaks are assayed forboth oligonucleotide and alkaline phosphatase activity and the productbearing fractions are combined. Typically the first peak will be excessenzyme, the second peak the oligonucleotide-protein conjugate and thethird peak unreacted oligonucleotide. Isolation of the product from theproduct-bearing fractions via HPLC and desalting on Sephadex G-25 willyield an oligonucleotide of the sequence: Oligomer 28: CTG TCT CCA* TCCTCT TCA CTwherein A* represents a nucleotide functionalized to incorporate analkaline phosphatase functionality linked via an2′-O-pentyl-amino-sulfo-SMCC(sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate)linking group to the 2′ position of the designated nucleotide.

EXAMPLE 10 Heterocyclic Base Peptide-Conjugated Oligonucleotides

Utilizing the method of Example 9-A-1 the intermediate amino linkeroligonucleotide of Example 2-A is reacted with sulfo-SMCC reagent. Theisolated oligonucleotide-maleimide conjugate is then further reactedwith SV40 peptide as per Example 9-A-2. This will give anoligonucleotide of the structure: Oligomer 29: TTG CTT* CCA TCT TCC TCGTCwherein T* represents a nucleotide functionalized to include a peptidelinked via an extended linker to the heterocyclic base of a2′-deoxyuridine nucleotide.

EXAMPLE 11 3′-Terminus Prot in-Conjugated oligonucleotides

Utilizing the method of Example 9-B-1 the 2′-O-methyl derivatizedintermediate amino linker oligonucleotide of Example 6-A is reacted withDSS reagent. The isolated oligonucleotide-disuccinimidyl suberateconjugate is then further reacted with a lysine containing NucleaseRNase H using the method of Example 9-B-2. This will give anoligonucleotide of the structure: Oligomer 30: C_(S)C_(S)C_(S)A_(S)G_(S)G_(S) C_(S)U_(S)C_(S) A_(S)G_(S)A-3′-proteinwherein protein represents RNase H, the subscript “is” represents aphosphorothioate inter-nucleotide backbone linkage and each of thenucleotides of the oligonucleotide includes a 2′-O-methyl group thereon.

EXAMPLE 12 5′-Terminus Protein-Conjugated 2′-O-Methyl DerivatizedOligonucleotides

Utilizing the method of Example 9-B-1 the 2′-O-methyl derivatizedintermediate amino linker oligonucleotide of Example 6-A (Oligomer 6) isreacted with DSS reagent. The isolated oligonucleotide-disuccinimidylsuberate conjugate is then further reacted with a lysine containingStaphylococcal Nuclease using the method of Example 9-B-2. This willgive an oligonucleotide of the structure: Oligomer 31:5′-protein-C_(S)C_(S)C_(S) A_(S)G_(S)G_(S) C_(S)U_(S)C_(S) A_(S)G_(S)A3′wherein protein represents Staphylococcal Nuclease, the subscript “s”represents a phosphorothioate inter-nucleotide backbone linkage and eachof the nucleotides of the oligonucleotide includes a 2′-O-methyl groupthereon.

Procedure A Confirmation of Structure of Functionalized OligonucleotidesContaining a Tethered 2′-Amino Moiety

Oligonucleotides of the invention were digested with snake venomphosphodiesterase and calf-intestine alkaline phosphatase to theirindividual nucleosides. After digestion, the nucleoside composition wasanalyzed by HPLC.

The HPLC analysis established that functionalized nucleotide compoundshaving the tethered 2′-amino moiety thereon were correctly incorporatedinto the oligonucleotide.

Snake venom phosphodiesterase [Boehringer-Mannheim cat. #108260, 1 mg(1.5 units)/0.5 ml] and alkaline phosphatase from calf intestine (1unit/microliter, Boehringer-Mannheim cat. # 713023) in Tris-HCl buffer(pH 7.2, 50 mM) were used to digest the oligonucleotides to theircomponent nucleosides. To 0.5 O.D. units of oligonucleotide in 50 μlbuffer (nearly 40 μM final concentration for a 20 mer) was added 5 μl ofsnake venom phosphodiesterase (nearly 0.3 units/mL, final concentration)and 10 μl of alkaline phosphatase (app. 150 units/mL, finalconcentration). The reaction mixture was incubated at 37° C. for 3hours. Following incubation, the reaction mixture was analyzed by HPLCusing a reverse phase analytical column (app. 30×2.5 cm); solvent A: 50mM TEAA pH 7; solvent B: acetonitrile; gradient 100% for 10 mins, then5% B for 15 mins, then 10% B and then wash. The results of thesedigestion are shown in Table 2 for representative oligonucleotides.TABLE 2 OLIGONUCLEOTIDE ANALYSIS VIA ENZYMATIC DIGESTION ObservedRatios** Abs. max. 267 252 267 260 Oligomer C G T A* A Oligomer 10 2 1 1Oligomer 11 3 5 2 1 Oligomer 12 9 1 8 1 1 Oligomer 13 9 1 8 2*Nucleoside having 2′-O-linker attached thereto; and**Corrected to whole numbers.As is evident from Table 2, the correct nucleoside ratios are observedfor the component nucleotides of the test oligonucleotides.

Procedure B Determination of Melting Temperatures (Tm's) of Cholic AcidOligonucleotide Conjugates

The relative ability of oligonucleotides to bind to their complementarystrand is compared by determining the melting temperature of thehybridization complex of the oligonucleotide and its complementarystrand. The melting temperature (Tm), a characteristic physical propertyof double helices, denotes the temperature in degrees centigrade atwhich 50% helical versus coil (un-hybridized) forms are present. Tm ismeasured by using the UV spectrum to determine the formation andbreakdown (melting) of hybridization. Base stacking, which occurs duringhybridization, is accompanied by a reduction in UV absorption(hypochromicity). Consequently a reduction in UV absorption indicates ahigher T_(m). The higher the Tm, the greater the strength of the bindingof the strands. Non-Watson-Crick base pairing has a strong destabilizingeffect on the Tm. Consequently, absolute fidelity of base pairing isnecessary to have optimal binding of an antisense oligonucleotide to itstargeted RNA.

1. Terminal End Conjugates

a. Synthesis

A series of oligonucleotides were synthesized utilizing standardsynthetic procedures (for un-functionalized oligonucleotides) or theprocedure of Example 3-A above for oligonucleotides having a 5′-terminusamino linker bearing oligonucleotide or the procedure of Example 3-B for5′-terminus cholic acid-bearing oligonucleotides. Each of theoligonucleotides had the following 5-LO antisense sequence: 5′ TCC AGGTGT CCG CAT C 3′. The nucleotides were synthesized on a 1.0 μmol scale.Oligomer 32 was the parent compound having normal phosphodiesterinter-nucleotide linkages. Oligomer 33 incorporated phosphorothioateinter-nucleotide linkages in the basic oligonucleotide sequence.Oligomer 34 is a an intermediate oligonucleotide having a 5′-aminolinkat the 5′-terminus of the basic oligonucleotide sequence and Oligomer 35was a similar 5′-aminolink compound incorporating phosphorothioateinter-nucleotide linkages. Oligomer 36 is a 5′-terminus cholic acidconjugate of the basic phosphodiester oligonucleotide sequence whileOligomer 37 is a similar 5′-cholic acid conjugate incorporatingphosphorothioate inter-nucleotide linkages. Oligomers 32 and 33 weresynthesized in a “Trityl-On” mode and were purified by HPLC. Oligomers34 and 35 were synthesized as per Example 30-A above without or withBeaucage reagent treatment, to yield phosphodiester or phosphorothioateinter-nucleotide linkages, respectively. Oligomers 36 and 37 wereprepared from samples of oligomers 34 and 35, respectively, utilizing asolution of cholic acid N-hydroxysuccinimide ester (Compound 1) 1dissolved in DMF as per Example 3-B. Oligomers 36 and 37 were purifiedby HPLC. The products were concentrated and desalted in a Sephadex G-25column. Gel electrophoresis analyses also confirmed a pure product withthe pure conjugate moving slower than the parent oligonucleotide or5′-amino functionalized oligonucleotide.

b. Melting Analysis

The test oligonucleotides [either the phosphodiester, phosphorothioate,cholic acid conjugated phosphodiester, cholic acid conjugatedphosphorothioate or 5′-aminolink intermediate phosphodiester orphosphorothioate oligonucleotides of the invention or otherwise] andeither the complementary DNA or RNA oligonucleotides were incubated at astandard concentration of 4 uM for each oligonucleotide in buffer (100mM NaCl, 10 mM Na-phosphate, pH 7.0, 0.1 mM EDTA). Samples were heatedto 90 degrees C. and the initial absorbance taken using a GuilfordResponse II spectro-photometer (Corning). Samples were then slowlycooled to 15 degrees C. and then the change in absorbance at 260 nm wasmonitored during the heat denaturation procedure. The temperature waselevated 1 degree/absorbance reading and the denaturation profileanalyzed by taking the 1st derivative of the melting curve. Data wasalso analyzed using a two-state linear regression analysis to determinethe Tm's. The results of these tests are shown in Table 3 as are theHPLC retention times of certain of the test compounds. TABLE 3 MeltingTemperature Of The Hybridization Complex Of The Oligonucleotide And ItsComplementary Strand HPLC Ret. Tm** Time* Oligomer DNA RNA minutes 3262.6 62.0 — 33 55.4 54.9 — 34 ND ND 13.6 35 ND ND 17.0 36 63.4 62.4 22.037 56.3 55.8 22.5*HPLC conditions: Walters Delta Pak C-18 RP 2.5 u column; at 0 min 100%0.1 TEAA; at 30 min 50% TEAA and 50% Acetonitrile: Flow rate 1.0 ml/min.**Tm at 4 μM each strand from fit of duplicate melting curves to 2-statemodel with linear sloping base line. Conditions: 100 mM NaCl, 10 mMPhosphate, 0.1 mM EDTA, pH 7.0.ND = not determined

As is evident from Table 2, conjugates of cholic acid at the end of theoligonucleotide do not affect the Tm of the oligonucleotides.

2. Strands Incorporating 2′-O-Pentylamino Linker

a. Synthesis

An oligonucleotide of the sequence: Oligomer 38: GGA* CCG GA*A* GGT A*CGA*G

wherein A* represents a nucleotide functionalized to incorporate apentylamino functionality at its 2′-position was synthesized in a onemicromole scale utilizing the method of Example 7-B. The oligonucleotidewas purified by reverse phase-HPLC, detritylated and desalted onSephadex G-25. PAGE gel analysis showed a single band. A furtheroligonucleotide, Oligomer 39, having the same sequence but without any2′-O-amino linker was synthesis in a standard manner. A complementaryDNA oligonucleotide of the sequence: Oligomer 40: CCT GGC CTT CCA TGCTC.

was also synthesized in a standard manner as was a complementary RNAoligonucleotide of the sequence: Oligomer 41: CCU GGC CUU CCA UGC UC

b. Melting Analysis

Melting analysis was conducted as per the method of Procedure B-1-b. Theresults are shown in Table 4. TABLE 4 Melting Temperature Of TheHybridization Complex Of The Oligonucleotide And Its ComplementaryStrand Tm* Oligomer DNA¹ RNA² 38 54.5 58.0 39 60.6 56.9*Tm at 4 μM each strand from fit of duplicate melting curves to 2-statemodel with linear sloping base line. Conditions: 100 mM NaCl, 10 mMPhosphate, 0.1 mM EDTA, pH 7.0.¹Against DNA complementary strand, Oligomer 40.²Against RNA complementary strand, Oligomer 41

As is evident from Table 4 against the RNA complementary strand thechange in Tm's between the strand having 2′-amino linkers thereon andthe unmodified strand is 1.1 degrees (0.22 change per modification).Against the DNA strand, the change is −6.1 degrees (−1.2 change permodification). When compared to the parent unmodified oligonucleotidethe 2′-amino linker-containing strand has a stabilizing effect uponhybridization with RNA and a destabilizing effect upon hybridizationwith DNA.

Compounds of the invention were tested for their ability to increasecellular uptake. This was determined by judging either their ability toinhibit the expression of bovine papilloma virus-1 (BPV-1) or an assayinvolving luciferase production (for HIV-1).

Procedure C Determination of Cellular Uptake Judged by the Inhibition ofExpression of Bovine Papilloma Virus-1 (bpv-1) as Measured by an E2Transactivation Assay

For this test, a phosphorothioate oligonucleotide analog of thesequence: Oligomer 42: CTG TCT CCA TCC TCT TCA CTwas used as the basic sequence. This sequence is designed to becomplementary to the translation initiation region of the E2 gene ofbovine papilloma virus type 1 (BPV-1). Oligomer 42 served as thepositive control and standard for the assay. Oligomer 3 (from Example 4above) served as a second test compound. It has the same basic sequenceexcept it is a phosphorothioate oligonucleotide and further it has acholic acid moiety conjugated at the 3′-end of the oligonucleotide.Oligomer 2 (from Example 2 above) served as a third test compound. Againit is of the same sequence, it is a phosphorothioate oligonucleotide andit has a cholic acid moiety conjugated at the 5′-end. Oligomer 5 (fromExample 5 above) served as a fourth test compound. Once again it has thesame sequence, is a phosphorothioate oligonucleotide and it has a cholicacid moiety conjugated at both the 3′-end and 5′-end. A fifth testcompound was a phosphorothioate oligonucleotide with no significantsequence homology with BPV-1. A sixth test compound was a furtherphosphorothioate oligonucleotide with no significant sequence homologywith BPV-1. The last test compound, the seventh test compound, was aphosphorothioate oligonucleotide with cholic acid conjugated to the3′-end but having no significant sequence homology with BPV-1. Compoundsfive, six and seven served as negative controls for the assay.

For each test I-38 cells were plated at 5×10⁴ cells per cm² in 60 mmpetri dishes. Eight hours after plating, medium was aspirated andreplaced with medium containing the test oligonucleotide and incubatedovernight. Following incubation, medium was aspirated and replaced withfresh medium without oligonucleotide and incubated for one hour. Cellswere then transfected by the CaPO₄ method with 2 ug of pE2RE-1-CAT.After a four hour incubation period cells were glycerol shocked (15%glycerol) for 1 minute followed by washing 2 times with PBS. Medium wasreplaced with DMEM containing oligonucleotide at the originalconcentration. Cells were incubated for 48 hours and harvested. Celllysates were analyzed for chloramphenicol acetyl transferase by standardprocedures. Acetylated and nonacetylated ¹⁴C-chloramphenicol wereseparated by thin layer chromatography and quantitated by liquidscintillation. The results are expressed as percent acetylation.

Two lots of the positive control compound were found to acetylate at alevel of 29% and 30%. The negative controls, test compounds five, sixand seven, were found to acetylate at 59%, 58% and 47%, respectively.The 3′-cholic acid conjugate test compound, Oligomer 3, was found toacetylate to 23%, the 5′-cholic acid conjugate test compound, Oligomer2, was found to acetylate to 36% and the test compound conjugated atboth the 3′-end and the 5′-end, Oligomer 5, was found to acetylate to27%.

The results of this test suggests that placement of a cholic acid moietyat the 3′-terminus of an oligonucleotide increase the activity. This inturn suggests that the increased activity was the result of increasedcellular membrane transport.

Procedure D Determination of Cellular Uptake Judged by Inhibition ofpHIV1uc with Cholic Acid Linked 2′-O-Methyl Substituted Oligonucleotides

For this test the absence of an oligonucleotide in a test well served asthe control. All oligonucleotides were tested as 2′-O-methyl analogs.For this test an oligonucleotide of the sequence: Oligomer 43: CCC AGGCUC AGA

where each of the nucleotides of the oligonucleotide includes a2′-O-methyl substituent group served as the basic test compound. Thesecond test compound of the sequence: Oligomer 44: 5′-CHA CCC AGG CUCAGA

wherein CHA represents cholic acid and where each of the nucleotides ofthe oligonucleotide includes a 2′-O-methyl substituent group, was alsoof the same sequence as the first test compound. This second testcompound included cholic acid conjugated to its 5′-end and was preparedas per the method of Example 3 utilizing 2′-O-methyl phosphoramiditeintermediates as identified in Example 7-C. The third test compound ofthe sequence: Oligomer 45: CCC AGG CUC AGA 3′-CHA

wherein CHA represents cholic acid and where each of the nucleotides ofthe oligonucleotide includes a 2′-O-methyl substituent group was also ofthe same sequence as the first test compound. The third test compoundincluded cholic acid conjugated to its 3′-end and was prepared as perthe method of Example 4 utilizing 2′-O-methyl phosphoramiditeintermediates as identified in Example 7-C. The fourth test compound wasa 2′-O-Me oligonucleotide of a second sequence: Oligomer 46: GAG CUC CCAGGC

where each of the nucleotides of the oligonucleotide includes a2′-O-methyl substituent group. The fifth test compound was of sequence:Oligomer 47: 5′-CHA GAG CUC CCA GGC.

wherein CHA represents cholic acid and where each of the nucleotides ofthe oligonucleotide includes a 2′-O-methyl substituent group. It was ofthe same sequence as the fifth test compound. This test compoundincluded cholic acid conjugated to its 5′-end and was prepared as perthe method of Example 3 utilizing 2′-O-methyl phosphoramiditeintermediates as identified in Example 7-C. A sixth test compound was arandomized oligonucleotide of the sequence: Oligomer 48: CAU GCU GCAGCC.

HeLa cells were seeded at 4×10⁵ cells per well in 6-well culture dishes.Test oligonucleotides were added to triplicate wells at 1 μM and allowedto incubate at 37° C. for 20 hours. Medium and oligonucleotide were thenremoved, cells washed with PBS and the cells were CaPO₄ transfected.Briefly, 5 μg of pHIVluc, a plasmid expressing the luciferase cDNA underthe transcriptional control of the HIV LTR constructed by ligating theKpnI/HindIII restriction fragments of the plasmids pT3/T7luc and pHIVpap(NAR 19(12)) containing the luciferase cDNA and the HIV LTRrespectively, and 6 μg of pcDEBtat, a plasmid expressing the HIV tatprotein under the control of the SV40 promoter, were added to 500 μl of250 mM CaCl₂, then 500 μl of 2×HBS was added followed by vortexing.After 30 minutes, the CaPO₄ precipitate was divided evenly between thesix wells of the plate, which was then incubated for 4 hours. The mediaand precipitate were then removed, the cells washed with PBS, and fresholigonucleotide and media were added. Incubation was continuedovernight. Luciferase activity was determined for each well thefollowing morning. Media was removed, then the cells washed 2× with PBS.The cells were then lysed on the plate with 200 μl of LB (1% Trit X-100,25 mM Glycylglycine pH 7.8, 15 mM MgSO₄, 4 mM EGTA, 1 mM DTT). A 75 μlaliquot from each well was then added to a well of a 96 well plate alongwith 75 μl of assay buffer (25 mM Glycylglycine pH 7.8, 15 mM MgSO₄, 4mM EGTA, 15 mM KPO₄, 1 mM DTT, 2.5 mM ATP). The plate was then read in aDynatec multiwell luminometer that injected 75 μl of Luciferin buffer(25 mM Glycylglycine pH 7.8, 15 mM MgSO₄, 4 mM EGTA, 4 mM DTT, 1 mMluciferin) into each well, immediately reading the light emitted (lightunits).

The random sequence compound (Oligomer 48) and the other non-cholicacid-conjugated test compounds (Oligomers 43 and 46) had comparableactivity. The 5′-conjugate of the first sequence (Oligomer 44) also hadactivity comparable to the non-conjugated compounds. The 5′-conjugate ofthe second sequence (Oligomer 47) showed a three-fold increase inactivity compared to the non-conjugated compounds and the 3′-conjugateof the first sequence (Oligomer 45) showed a further 3-fold increase inactivity compared to Oligomer 47.

All the test cholic acid-bearing oligonucleotides showed significantinhibition of luciferase production compared to non-cholic acid-bearingoligonucleotides. This suggests that the increased activity was theresult of increased cellular membrane transport of the cholicacid-bearing test oligonucleotides.

EXAMPLE 13 Retinoic Acid Conjugated Oligonucleotide

A. Retinoic Acid N-Hydroxysuccinimide Ester

Anhydrous DMF (150 ml) was added to a mixture of retinoic acid (15 mmol,4.5 g, Fluka) and N-hydroxysuccinimide (5.25 g, 45 mmol). The mixturewas stirred in the presence of argon. EDAC[ethyl-3-(3-dimethylamino)propyl carbodiimide] (4 ml, 25 nmol) was thenadded and this mixture was then stirred overnight. The solution was thenevaporated to a yellow gum and dissolved in 200 ml ethylacetate andwashed successively with 4% NaHCO₃ solution (200 ml) followed bysaturated NaCl solution, dried over anhydrous MgSO₄ and evaporated toyield the desired compound as a yellow solid in nearly 90% yield.

B. Retinol Phosphoramidite

All-trans-retinol (1 g) was vacuum dried and dissolved in anhydrousCH₂Cl₂ (10 ml) in an argon atmosphere. Diisopropylethylamine (2.65 ml,21.5 mmol) was syringed in and the reaction mixture was cooled in anice-bath. 2-cyanoethyl-N,N-diisopropylchlorophosphoramidate (2.5 g, 2.45ml, 10.5 mmol) was slowly added by syringe under argon atmosphere. Thereaction mixture was stirred for 30 min. at which time TLC(CH₂Cl₂:CH₃OH:Et₃N, 90:10:0.1) indicated complete conversion of thealcohol to its phosphoramidite. The reaction mixture was added to 100 mlof saturated NaHCO₃ followed by washing the reaction flask with (2×25ml) CH₂Cl₂. The CH₂Cl₂ layer was separated and washed with 100 ml ofsaturated NaCl solution and dried over anhydrous MgSO₄ and evaporatedinto a yellow foam. The amidite was used directly in the DNA synthesizerwithout further purification since decomposition of this amidite wasnoted upon silica column purification.

C. Retinoic Acid Functionalized Oligonucleotide

Multiple batches of an oligonucleotide of the sequence: Oligomer 49:T_(s)G_(s)G_(s) G_(s)A_(s)G_(s) C_(s)C_(s)G_(s) T_(s)A_(s)G_(s)C_(s)G_(s)A_(s) G_(s)G_(s)C_(s)-3′ALwherein AL represents a 3′-aminolinker and “s” represents aphosphorothioate inter-nucleotide backbone linkage were synthesized asper the procedure of Example 4 on 10 μmol scales in the standard manneron the DNA synthesizer utilizing phosphoramidite methodology employing3′-amine-ON solid support available from Clontech. During the synthesis,the phosphorothioate backbone was formed by the Beaucage reagent. Theoligonucleotide was deprotected and purified using standard protocols.

The 3′-aminolinker-oligonucleotide (oligomer 49, 100 OD units,approximately 550 nmols) was dissolved in freshly prepared NaHCO3 buffer(500 μl, 0.2M, pH 8.1) and treated with a solution-of retinoic acidN-hydroxy succinimide ester (50 mg, 125 μmols) dissolved in 500 μl ofDMF. The reaction mixture was covered with aluminum foil and left at 37°C. bath overnight. It was then passed through a Sephadex G-25 column(1×40 cm) and the first eluant was collected, concentrated and passedagain through another Sephadex G-25 column (1×40 cm) to remove excessVitamin-A reagent. Concentration of the yellow oligonucleotide fractionsfollowed by HPLC purification yielded the retinoic acid-oligonucleotideconjugate.

EXAMPLE 14 Folic Acid Conjugated Oligonucleotide

A mixture of folic acid (30 mg, 68 μmols) and 1-hydroxybenzotriazole (30mg, 222 μmols) was dissolved in 900 ml of dry DMF. To this solution, 50ml of EDAC (312 μmols) was added. The resultant yellow viscous materialwas vortexed well and 500 ml from the solution was transferred into 100O.D. units of the 3′-aminolinker oligonucleotide (Oligomer 49, 537μmols) dissolved in 0.2M NaHCO₃ buffer. The yellow solution wasvortexed, covered with aluminum foil and allowed to react for 16 hrs.The mixture was then loaded into a Sephadex G-25 column (1×40 cm). Theoligonucleotide fraction was collected, concentrated and passed one moretime through Sephadex G-25 column. The oligonucleotide fractions wereconcentrated and purified by reverse phase HPLC. The conjugate appearedas a broad peak centered around 32.5 min. while the oligonucleotidestarting material had a retention time of 30 min. (5%→40% of 100% CH₃CNover 60 min. as solvent B and 50 mM TEAA pH 7.0 as the solvent A inreverse phase HPLC). The conjugate was concentrated and passed throughSephadex G-25 column again for desalting. Gel analysis indicated aslower moving material than the starting oligonucleotide.

EXAMPLE 15 Methyl Folate Conjugated Oligonucleotide

In a like manner to Example 14, 5-methyl folate was also attached toOligomer 49.

EXAMPLE 16 Pyridoxal Conjugated Oligonucleotide

The 3′-aminolinker-oligonucleotide (Oligomer 49, 20 O.D. units,approximately 110 nmols, based on the calculated extinction coefficientof 1.828×10⁵ at 260 nm) was dissolved in 100 microliters of water. 100ml of 1M NaOAc buffer (pH 5.0) was added followed by 5 mg of pyridoxalhydrochloride (24 μmols) and 50 μl of 60 mM NaCNBH₃ solution. Thesolution was vortexed and left aside for overnight. It was then passedthrough a Sephadex G-25 column and further purified in an analyticalHPLC column.

EXAMPLE 17 Tocopherol Conjugated Oligonucleotide

A. Vitamin E (Tocopherol)-hemisuccinate-NHS ester

α-Tocopherolhemisuccinate (Sigma, 5 g, 9.4 mmols) was treated with 3equivalents of N-hydroxysuccinimide and 2 equivalents of EDAC asdescribed under the vitamin-A NHS ester synthesis, Example 13-A above.Work up in the same manner as Example 13 yielded the title compound as alight brown wax-like solid.

B. Tocopherol Conjugated Oligonucleotide

α-Tocopherol-hemisuccinate-NHS ester was treated with Oligomer 49 in thesame manner described in Example 13-C for the retinoic acid conjugation.The conjugate was obtained in nearly 50% yield.

EXAMPLE 18 Synthesis of Uridine Based Aminolinkers

A. Preparation of 5′-dimethoxytrityl-2′-(O-pentyl-N-phthalimido)uridinephosphoramidite

Utilizing the protocol of Wagner, et al., J. Org. Chem. 1974, 39, 24,uridine (45 g, 0.184 mol) was refluxed with di-n-butyltinoxide (45 g,0.181 mol) in 1.4 1 of anhydrous methanol for 4 hrs. The solvent wasfiltered and the resultant 2′,3′-O-dibutylstannylene-uridine was driedunder vacuum at 100° C. for 4 hrs to yield 81 g (93%).

The 2′,3′-O-dibutyl stannylene-uridine was dried over P₂O₅ under vacuumfor 12 hrs. To a solution of this compound (20 g, 42.1 mmols) in 500 mlof anhydrous DMF were added 25 g (84.2 nmols) ofN(5-bromopentyl)phthalimide (Trans World Chemicals, Rockville, Md.) and12.75 g (85 mmols) of cesium fluoride (CeF) and the mixture was stirredat room temperature for 72 hrs. The reaction mixture evaporated,coevaporated once with toluene and the white residue was partitionedbetween EtOAc and water (400 ml. each). The EtOAC layer was concentratedand applied to a silica column (700 g). Elution with CH₂Cl₂—CH₃OH (20:1v/v) gave fractions containing a mixture of the 2′- and 3′-isomers ofO-pentyl-ω-N-phthalimido uridine, in 50% yield.

The mixture was allowed to react with DMT chloride in dry pyridine atroom temperature for 6 hrs. CH₃OH was used to quench excess DMT-Cl andthe residue was partitioned between CH₂Cl₂ containing 0.5% Et₃N andwater. The organic layer was dried (MgSO₄) and the residue was appliedto a silica column. Elution with CH₂Cl₂:CH₃OH (20:1, v/v) separated the2′ and 3′ isomers.

The 2′-O-pentyl-ω-N-phthalimido-5′-DMT-uridine was converted to itsphosphoramidite as per the procedure referenced in Example 7.

B. Preparation of 5′-dimethoxytrityl-2-(O-hexyl-N-phthalimido)uridinephosphoramidite

In a like manner to Example 18-A, using N-(6-bromohexyl) phthalimide, a2′-six carbon aminolinker was introduced at the 2′-position of uridine.

C. Preparation of 5′-dimethoxytrityl-2-(O-decyl-N-phthalimido)uridinephosphoramidite

In a like manner to Example 18-A N-(10-bromodecyl)phthalimide wassimilarly used to introduce a 2′-ten carbon aminolinker in thenucleotide.

EXAMPLE 19 Synthesis of Cytidine Based Aminolinkers

A. Preparation of 5′-dimethoxytrityl-2-(O-propyl-N-phthalimido)cytidinephosphoramidite

The 5′-DMT protected 2′-O-functionalized cytidine phosphoramidite wasprepared as per the procedure of Example 7 substituting cytidine foradenosine.

B. Preparation of Oligonucleotides Having a 2′-Aminolinker Bearing3′-Terminal Nucleotide

The following oligonucleotides having phosphodiester inter-nucleotidelinkages and a 2′-aminolinker at the 3′ terminal nucleotide weresynthesized: Oligomer 50: GGC GUC UCC AGG GGA UCU GAC* Oligomer 51: TCTGAG TAG CAG AGG AGC TC*wherein C* represents a nucleotide functionalized to incorporate apropyl-N-phthalimido functionality. Oligomer 50 is antisense to the Capregion of CMV and Oligomer 51 is antisense to an ICAM sequence. Theoligonucleotides were synthesized on a 3 μmol scale. Upon completion ofsynthesis they were deprotected using standard protocols and purified byreverse phase HPLC, detritylated and desalted.

EXAMPLE 20 Conversion of an Oligonucleotide Having A 2′-Aminolinker Toan Oligonucleotide Having A Thiolinker

A. Oligomer 50

Oligomer 50 (25 O.D. units) was treated with 5 mg SATA(N-succinimidyl-S-acetylthioacetate) in 0.2M NaHCO₃ buffer. The reactionmixture was passed through a Sephadex G-25 column, the oligonucleotidefraction was concentrated and treated with 200 mM NH₂OH hydrochloridesolution in water (1 ml).

B. Oligomer 51

Oligomer 51 (25 O.D. units) was treated with 5 mg SATA(N-succinimidyl-S-acetylthioacetate) in 0.2M NaHCO₃ buffer. The reactionmixture was passed through a Sephadex G-25 column, the oligonucleotidefraction was concentrated and treated with 200 mM hydroxylaminehydrochloride solution in water (1 ml).

EXAMPLE 21 Conjugation of o-Phenanthroline at 2′-Position ofOligonucleotides

A. Oligomer 52

To the solution resulting from Example 20-A was added 2 mg of5-(iodoacetamide)-o-phenanthroline reagent followed by shakingovernight. The conjugate was purified by a size exclusion column andreverse phase HPLC to yield Oligomer 52: GGC GUC UCC AGG GGA UCUGAC-2′PHAwherein PHA represents a nucleotide functionalized at its 2′-positionwith phenanthroline via a thiol linker of the structure2′-O—(CH₂)₃—NH—C(═O)—CH₂—S—CH₂—C(═O)—NH—.

B. Oligomer 53

To the solution resulting from Example 20-A was added 2 mg of5-(iodoacetamide)-O-phenanthroline reagent followed by shakingovernight. The conjugate was purified by a size exclusion column andreverse phase HPLC to yield Oligomer 53: TCT GAG TAG CAG AGG AGCTC-2′PHAwherein PHA represents a nucleotide functionalized at its 2′-positionwith phenanthroline via a thiol linker of the structure2′-O—(CH₂)₃—NH—C(═O)—CH₂—S—CH₂—C(═O)—NH—.

EXAMPLE 22 Oligonucleotide Having a Nucleotide with aCrosslinker/Alkylator Attached Via a 2′-Aminolinker in a InternalPosition in the Oligonucleotide

A. Synthesis of an Oligonucleotide Having a Uridine 2′-Aminolinker

The following oligonucleotide having phosphodiester inter-nucleotidelinkages and a 2′-aminolinker at an internal position is synthesizedutilizing the uridine 2′-aminolinker of Example 18: Oligomer 54: GGC CAGAUC UGA GCC UGG GAG CU*C UGU GGC Cwherein U* represents a nucleotide functionalized to incorporate apropyl-N-phthalimido functionality. Oligomer 50 is an oligonucleotidecorresponding to positions G₁₆ to C₄₆ of TAR RNA.

B. Conjugation of Iodo Acetamide to U₃₈ Position of TAR Structure

Oligomer 54 is reacted with iodoacetic acid N-hydroxysuccinimide esterto form the iodoacetamide derivative at the U₃₈ position of the TARstructure. The U₃₈ position is thus available for crosslinking to the 7position of the guanine base of either G₂₆ or G₂₈ of the TAR structure.

EXAMPLE 23 Conjugation of Pyrene at 2′-Position of Oligonucleotides

A. Single 2′ Site Modification

10 O.D. units (A₂₆₀) of Oligomer 12 (Example 7-B) (approximately 60nmols based on the calculated extinction coefficient of 1.68×10⁵) wasdried in a microfuge tube. It was dissolved in 200 μml of 0.2 M NaHCO₃buffer and pyrene-1-butyric acid N-hydroxysuccinimide ester (i.e.,succinimidyl-1-pyrene butyrate, 3 mg, 7.79 μmols, Molecular Probes,Eugene, Oreg.) was added followed by 400 μl of DMF. The mixture wasincubated at 37° C. overnight. The solution was applied to a SephadexG-25 column (1×40 cm) and the oligonucleotide fractions were combined.The product was purified by HPLC. The pyrene conjugates exhibited thetypical pyrene absorption between 300 and 400 nm. The product had a HPLCretention time of 26.94 min. while the parent oligonucleotide had aretention time of 21.78 min. (Waters 600E with 991 detector; HamiltonPRP-1 column (15×25 cm); Solvent A: 50 mM TEAA, pH 7.0; B: 45 mM TEAAwith 80% Acetonitrile; 1.5 mL/min. flow rate: Gradient 5% B for first 5minutes, linear (1%) increase in B every minute afterwards).

B. Multiple 2′ Site Modifications

10 O.D. units of Oligomer 12 (Example 7-B) was treated with twice theamount of pyrene-1-butyric acid N-hydroxysuccinimide (6 mg in 400 μlDMF) and worked up in the same fashion as Example 23-A. Sephadex G-25purification followed by HPLC purification gave the doublypyrene-conjugated oligonucleotide. The doubly conjugated oligonucleotideexhibited a HPLC retention time of 32.32 min. while the parentoligonucleotide had a retention time of 21.78 min. (Waters 600E with 991detector; Hamilton PRP-1 column (15×25 cm); Solvent A: 50 mM TEAA, pH7.0; B: 45 mM TEAA with 80% Acetonitrile; 1.5 mL/min. flow rate:Gradient 5% B for first 5 minutes, linear (1%) increase in B everyminute afterwards).

EXAMPLE 24 Conjugation of Acridine at 2′-Position of Oligonucleotides

A. Single 2′ Site Modification

10 O.D. units (A₂₆₀) of Oligomer 12 (Example 7-B, about 60 nmols) wasdried and dissolved in 1M NaHCO₃/Na2CO₃ buffer, pH 9.0, 200 μl.9-acridinyl-isothiocyante, (5 mg, 2.1 μmols, Molecular Probes, Eugene,Oreg.) was dissolved in 200 μl DMF. This solution was added to theoligonucleotide, vortexed, covered with aluminum foil and left at 37° C.overnight. The reaction mixture was purified by passing through aSephadex G-25 column (1×40 cm) concentrated and further purified by HPLC(reverse-phase). The product had a HPLC retention time of 25.32 min.while the parent oligonucleotide had a retention time of 21.78 min.(Waters 600E with 991 detector; Hamilton PRP-1 column (15×25 cm);Solvent A: 50 mM TEAA, pH 7.0; B: 45 mM TEAA with 80% Acetonitrile; 1.5mL/min. flow rate: Gradient 5% B for first 5 minutes, linear (1%)increase in B every minute afterwards).

B. Multiple 2′ Site Modifications

10 O.D. units (A₂₆₀) of Oligomer 13 (Example 7-B) in 400 μl of 1MNa2CO₃/NaHCO₃ buffer (pH 9.0) was treated with 10 mg of9-acridinyl-isothiocyanate in 400 μl of DMF. The reaction mixture wasvortexed, covered with aluminum foil and left at 37° C. overnight. Thereaction mixture was purified as for the single site reaction of Example24-A. The doubly conjugated acridine-oligonucleotide eluted as the lastpeak in the HPLC following single-modification products. The product hada HPLC retention time of 32.32 min. while the parent oligonucleotide hada retention time of 21.78 min. (Waters 600E with 991 detector; HamiltonPRP-1 column (15×25 cm); Solvent A: 50 mM TEAA, pH 7.0; B: 45 mM TEAAwith 80% Acetonitrile; 1.5 mL/min. flow rate: Gradient 5% B for first 5minutes, linear (1%) increase in B every minute afterwards).

EXAMPLE 25 Conjugation of Porphyrin at 2′-Position of Oligonucleotides

Methylpyroporphyrin XX1 ethyl ester (Aldrich) is condensed withaminocaproic acid using N-hydroxysuccinimide and EDAC. The resultantcarboxylic acid is then activated again with N-hydroxy succinimide andEDAC and treated with Oligomer 12 as per the procedure of Example 23-Ato give the 2′-porphyrin conjugated oligonucleotide.

EXAMPLE 26 Conjugation of Hybrid Intercalator-Ligand at 2′-Position ofOligonucleotides

A. Photonuclease/Intercalator Ligand

The photonuclease/intercalator ligand6-[[[9-[[6-(4-nitrobenzamido)hexyl]amino]acridin-4-yl]carbonyl]amino]hexanoyl-pentafluorophenylester was synthesized as per the procedure of Egholm et al., J. Am.Chem. Soc. 1992, 114, 1895.

B. Single 2′ Site Modification

10 O.D. units of Oligomer 12 (Example 7-B) was dissolved in 100 μl of0.1 M borate buffer (pH 8.4) and treated with 330 μl of DMF solution (10mg in 1 ml of DMF) of6-[[[9-[[6-(4-nitrobenzamido)hexyl]amino]acridin-4-yl]carbonyl]amino]hexanoylpentafluorophenylester. The solution was covered with aluminum foil and allowed to reactovernight. The product was purified by Sephadex G-25 and HPLCpurification of the reaction mixture.

C. Multiple 2′ Site Modification

10 O.D. units A₂₆₀ of Oligomer 13 (Example 7-B) was dissolved in 200 μlof 0.1 M borate buffer (pH 8.4) and treated with 660 μl of the DMFsolution of6-[[[9-[[6-(4-nitrobenzamido)hexyl]amino]acridin-4-yl]carbonyl]amino]hexanoylpentafluorophenylester (10 mg in 1 ml solution) and the solution was covered withaluminum foil and left aside overnight. The bright yellow solution waspurified by Sephadex G-25 and reverse phase HPLC to give the doublyconjugated oligonucleotide.

EXAMPLE 27 Conjugation of Bipyridine Complex at 2′-Position ofOligonucleotides

A. Bipyridine Complex

Succinimidyl-4-carboxyl-4′-methyl-2,2′-bipyridine is synthesizedaccording to the procedure of Telser, et al., J. Am. Chem. Soc. 1989,111, 7221.

B. Single 2′ Site Modification

10 O.D. A₂₆₀ units of Oligomer 12 is reacted with a 200 fold molarexcess of succinimidyl-4-carboxyl-4′-methyl-2,2′-bipyridine in 0.1 Mborate buffer pH 8.5/DMF. The solution is purified by Sephadex G-25 andreverse phase HPLC to yield the conjugated oligonucleotide.

EXAMPLE 28 Conjugation of Aryl Azide Photocrosslinkers at 2′-Position ofOligonucleotides

A. Conjugation of N-hydroxysuccinimidyl-4-azidobenzoate (HSAB)

Oligomer 14 (i.e., TTG CTT CCA* TCT TCC TCG TC wherein A* represents2′-O-pentyl amino adenosine, Example 7-C, 100 O.D. units, 595 nmols,based on the calculated extinction coefficient of 1.6792×10⁶ OD units)was dried and dissolved in 500 ml of 0.2M NaHCO₃ buffer pH 8.1 andtreated with 25 mg of N-hydroxysuccinimidyl-4-azidobenzoate (HSAB, 96μmols, available both from Pierce & Sigma)) dissolved in 500 μl of DMF.The reaction was allowed to react overnight at 37° C. and passed twiceover Sephadex G-25 column (1×40 cm). The oligonucleotide fraction waspurified by reverse-phase HPLC. The product had the HPLC retention timeof 38.79 min while the parent oligonucleotide had the retention time of33.69 min. (5% →40% CH3CN in 60 min.) in reverse phase column.

B. Conjugation ofN-succinimidyl-6(4′-azido-2′-nitrophenyl-amino)hexanoate

Oligomer 14 (Example 7-C, 200 OD units) was dissolved in 500 ml NaHCO₃buffer (0.2M, pH 8.1) and treated with 500 mg ofN-succinimidyl-6(4′-azido-2′-nitrophenyl-amino)hexanoate (SANPAH, 128μmols, available both from Pierce and Sigma) dissolved in 500 μl DMF.The reaction vial was wrapped with aluminum foil and heated at 37° C.overnight. The reaction mixture was passed twice over a Sephadex G-25column (1×40 cm). The oligonucleotide fraction was purified byreverse-phase HPLC. The product had the HPLC retention time of 40.69min. while the parent oligonucleotide had the retention time of 33.69min. (5%→40% CH₃CN in 60 min.) in a reverse phase column.

Procedure D Duplex Melting Temperature of Conjugated Oligonucleotides

Utilizing the protocol described in Procedure B-1-b, the meltingtemperatures of various of the 2-aminolinked conjugate oligomers of theinvention against a complementary DNA strand were obtained. Theconjugate oligomers were compared to an oligomer of the same sequencebearing a 2′-O-pentylamino group. An un-modified, i.e., wild type,strand of the same sequence was also tested for comparison purposes.Both single site and multiple site conjugated oligomers were tested. Asis shown in Table 5, the T_(m) and the ΔT_(m)/modification, both ascompared to 2′-pentylamino bearing oligomer, were measured. The wildtype sequence is: Oligomer 55: CTG TCT CCA TCC TCT TCA CT

the single site sequence is: Oligomer 12: CTG TCT CCA* TCC TCT TCA CT

and the multiple site sequence is: Oligomer 13: CTG TCT CCA* TCC TCTTCA* CT

where A* represents a site of conjugation. TABLE 5 Duplex MeltingTemperature of Conjugated Oligonucleotides Against DNA OligomerConjugate T_(m)° C. ΔT_(m)/mod 55 wild type 60.5 — 12 2′-O-pentyl-NH₂58.1 — 12 biotin 56.4 −1.7 12 cholic acid 55.5 −2.6 12 digoxigenin 55.8−2.3 12 fluorescein 55.1 −3.0 12 pyrene 62.6 +4.5 12 acridine 58.6 +0.513 2′-O-pentyl-NH₂ 56.9 — 13 biotin 54.4 −1.3 13 cholic acid 54.3 −1.313 digoxigenin 53.8 −1.6 13 fluorescein 53.4 −1.8 13 pyrene 65.1 +4.1 13acridine 58.1 +1.2

EXAMPLE 29 Conjugation of Imidazole-4-acetic acid at 2′-Position ofOligonucleotides

A. Activated imidazole-4-acetic acid

Imidazole-4-acetic acid was reacted with 2,4-dinitrofluoro benzene. Theresulting imidazole-N-(DNP)-4-acetic acid was converted to its N-hydroxysuccinimide ester by treating with NHS/EDAC as per the procedure ofExample 13.

B. 2′-Site Modification

10 O.D. A₂₆₀ units of oligomer 12 was reacted with a 100 fold molarexcess of imidazole-N-(DNP)-4-acetic acid NHS ester in 0.1M boratebuffer pH 8.5/DMF (200 μL each). After overnight reaction, the reactionmixture was treated with 200 μL of mercaptoethanol to cleave off the DNPprotecting group. The resulting reaction mixture was purified by passingthrough a Sephadex G-25 column followed by reverse phase HPLC to yieldthe imidazole conjugated oligonucleotide.

EXAMPLE 30 Conjugation of Metal Chelating Agents to 2′-Position ofOligonucleotide

A. EDTA Complex

To form an EDTA Fe(II) complex for coupling to an oligonucleotide as anucleic acid cleaving agent, tricylohexyl ester of EDTA is synthesizedaccording to the procedure of Sluka, et al., J. Am. Chem. Soc. 1990,112, 6369.

B. EDTA 2′-Site Modification

The tricyclohexyl ester of EDTA (1.25 mg, 1.94 mmol) andhydroxybenzotriazole (HOBt, 1 mg, 6.6 mmol) are dissolved in DMF (50 μL)and EDAC 10 μL is added. To this solution, oligonucleotide 12 (10 ODunits) in 100 μL g 0.1M borate buffer is added and left overnight. Thesolution is passed through a Sephadex G-25 column and theoligonucleotide fraction treated with conc. NH₃ (100 μL) for 1 hr. tocleave off the acid protecting groups. Finally purification is effectedby size exclusion and HPLC.

C. DTPA 2′-Site Modification

Oligomer 12 was treated with diethylene triamine pentaacetic anhydride(DTPA) in 0.1M NaHCO₃/DMF to offer single-site modification. Theconjugate was complexed with Gadolinium ion (Gd III) to give a contrastagent, usable among other uses, as an uptake measuring agent.

EXAMPLE 31 Conjugation of Cholesterol to the 2′-Position ofOligonucleotide

A. Method 1—2′-Aminolinker

Cholesterol-hemisuccinate was converted to its N-hydroxy succinimideester. It was then conjugated to Oligomer 12 as per the procedure ofExample 23-A or oligomer 13 as per the procedure of Example 23-B.

B. Method 2—Conjugation of Cholesterol Via a Disulfide Bridge

Step 1

Thiocholesterol (1.4 g,3.5 mmol) is added to a stirred solution of2,2′-dithiobis(5-nitropyridine) (1.4 g 4 mmol) in chloroform 20 mLcontaining glacial acetic acid (400 μL) under an argon atmosphere. Thereaction is allowed to continue overnight at room temperature, afterwhich the precipitated 5-nitropyridine-2-thione was removed and thesolution evaporated and purified in a silica column to giveS-(2-thio-5-nitropyridyl)-thio cholesterol.

Step 2 Oligomer 55: T_(s)G_(s)G_(s) G_(s)A_(s)G_(s) C_(s)C_(s)G_(s)T_(s)A*_(s)G_(s) C_(s)G_(s)A_(s) G_(s)G_(s)C_(s)wherein A* represents an adenosine nucleotide funtionalized toincorporate a 2′-O-pentylamino linking group is synthesized as perExample 7. This oligonucleotide is then converted into a thiol linkercompound as per the procedure described for Oligomer 50 in Example 20.

The thiol linker group containing oligonucleotide, oligomer 55, isreacted with an excess of S-(2-thio-5-nitropyridyl)-thiocholesterol toconjugate the cholesterol moiety to the 2′ position of theoligonucleotide via a disulfide bridge.

EXAMPLE 32 Synthesis of 2′-Aminolinker Containing Solid Supports forDNA/RNA Synthesis

A. 5′-Dimethyoxytrityl-2′-O-(pentyl-N-phthalimido)uridine

5′-Dimethyoxytrityl-2′-O-(pentyl-N-phthalimido)uridine was synthesizedas per the procedure described in Example 18.

B. Succinate Nucleoside

The nucleoside of step A (1 mmol) was treated with 4-DMAP (122 mg, 1mmol) and succinic anhydride (250 mg, 2.5 mmol) in 10 mL g anhydrouspyridine. After shaking overnight, TLC (EtOAc: Hexane 6:4 with 0.1%Et₃N) indicated complete succinylation of the nucleoside. 10 mL of waterwas added and the reaction shaken for an additional 1 hr. The reactionmixture was evaporated and partitioned between CHCl₃ and 20% citric acid(50 mL each). The chloroform layer was washed with brine and evaporated.It was then used in the next step.

C. Nitrophenyl Succinate

The dry 3′-O-succinate from step B was dissolved in dry dioxan (10 mL)containing pyridine (400 ml). 4-Nitrophenol (280 mg, 2 mmol) was addedfollowed by DCC (1.32 g, 5 mmol) and the solution was shaken for 24 hrs.The fine precipitate of urea was filtered and the filtrate evaporatedand applied to a silica column. The column was eluted with 5% CH₃OH inCHCl₃ containing 0.1% Et₃N. The 3′-nitrophenyl succinate of thenucleoside eluted first from the column. The product containingfractions were evaporated to give a foam.

D. Nucleoside Solid Support

The 3′-nitrophenyl succinate from step C was dissolved in 5 mL ofanhydrous DMF and treated with 5 g of 500 Å pore diameter aminopropylCPG support and shaken for 8 hrs. The CPG support was filtered, washedwith methanol (5×20 ml) followed by ether (5×20) and dried. The CPGsupport was capped by treating for 30 min. with pyridine/aceticanhydride/N-methyl imidazole (20 ml, 8:1:1 v/v/v). The CPG support wasthen filtered off, washed with pyridine, methanol and ether andair-dried. Assay of the dimethoxy trityl showed the loading capacity ofthe CPG support was 27 mmols/gram.

Procedure E

Determination of Cellular Uptake of Folic Acid ConjugatedOligonucleotide

The effect of conjugation of an oligonucleotide with folic acid wasdetermined by the inhibition of ICAM-1 utilizing the method of Chiang,et al., J. Biol. Chem. 1991, 266, 18162. Utilizing this method, humanlung epithelial carcinoma cells (A549 cells) were grown to confluence in96 well plates. Medium was removed and the cells were washed with folicacid free medium three times. Folic acid free medium was added to thecells and increasing concentrations of an ICAM-1 specific antisensephosphorothioate oligonucleotide having the sequence 5′-TGG GAG CCA TAGCGA GGC-3′, either free or conjugated to folic acid, was added to theincubation medium. This oligonucleotide is an 18 base phosphorothioateoligonucleotide that targets the AUG translation initiation codon of thehuman ICAM-1 mRNA (Chiang et al., J. Biol. Chem. 1991, 266, 18162). Theoligonucleotides were incubated with the A549 cells for 24 hours thenICAM-1 was induced by adding 2.5 ng/ml tumor necrosis factor-α to themedium. Cells were incubated an additional 15 hours in the presence oftumor necrosis factor-α and oligonucleotide. ICAM-1 expression wasdetermined by a specific ELISA as described by Chiang, et al. We hadpreviously demonstrated that the addition of the test oligonucleotide toincubation medium alone does not result in inhibition of ICAM-1expression. However formulation of the test oligonucleotide withcationic liposomes results in at least a 1000 fold increase in potencyand also correlates with the appearance of the oligonucleotide in thenucleus (Bennett, et al., Molecular Pharmacology 1991, 41, 1023). Theresults of this test are shown in Table 6. At the 3 uM level, the folicacid conjugated oligonucleotide shows an approximate 40% enhancement inactivity. TABLE 6 ICAM-1 Activity Oligonucleotide + FolicOligonucleotide Acid − Percent Concentration Control Of Control  1 uM105.1 107.6  3 uM 112.0 104.4 10 uM 112.4  92.9 30 uM 108.4  61.6

Those skilled in the art will appreciate that numerous changes andmodifications may be made to the preferred embodiments of the inventionand that such changes and modifications may be made without departingfrom the spirit of the invention. It is therefore intended that theappended claims cover all such equivalent variations as fall within thetrue spirit and scope of the invention.

1-44. Cancel.
 45. A compound comprising a plurality of linkednucleosides, wherein: at least one of the nucleosides is a2′-deoxy′-2′-fluoro, 2′-O—C₁-C₂₀-alkyl, 2′-O—C₂-C₂₀-alkenyl,2′-O—C₂-C₂₀-alkynyl, 2′-S—C₁-C₂₀-alkyl, 2′-S—C₂-C₂₀-alkenyl,2′-S—C₂-C₂₀-alkynyl, 2′-NH—C₁-C₂₀-alkyl, 2′-NH—C₂-C₂₀-alkenyl,2′-NH—C₂-C₂₀-alkynyl nucleoside; and at least one of the nucleosides isa 5′ terminal nucleoside having a non-aromatic lipophilic moleculelinked to the 5′-position of the nucleoside.
 46. The compound of claim45 wherein at least one of said nucleosides is a 2′-deoxy-2′-fluoro or a2′-methoxy nucleoside.
 47. A compound comprising a plurality of linkednucleosides, wherein: at least one of the nucleosides is a2′-deoxy′-2′-fluoro, 2′-O—C₁-C₂₀-alkyl, 2′-O—C₂-C₂₀-alkenyl,2′-O—C₂-C₂₀-alkynyl, 2′-S—C₁-C₂₀-alkyl, 2′-S—C₂-C₂₀-alkenyl,2′-S—C₂-C₂₀-alkynyl, 2′-NH—C₁-C₂₀-alkyl, 2′-NH—C₂-C₂₀-alkenyl,2′-NH—C₂-C₂₀-alkynyl nucleoside; and at least one of the nucleosides isa 5′ terminal nucleoside having a non-aromatic molecule linked to the5′-position of the nucleoside.
 48. The compound of claim 47 wherein atleast one of said nucleosides is a 2′-deoxy-2′-fluoro or a 2′-methoxynucleoside.
 49. A compound comprising a plurality of linked nucleosides,wherein: at least one of the nucleosides is a 2′-deoxy′-2′-fluoro,2′-O—C₁-C₂₀-alkyl, 2′-O—C₂-C₂₀-alkenyl, 2′-O—C₂-C₂₀-alkynyl,2′-S—C₁-C₂₀-alkyl, 2′-S—C₂-C₂₀-alkenyl, 2′-S—C₂-C₂₀-alkynyl,2′-NH—C₁-C₂₀-alkyl, 2′-NH—C₂-C₂₀-alkenyl, 2′-NH—C₂-C₂₀-alkynylnucleoside; and at least one of the nucleosides is a 5′ terminalnucleoside having a lipophilic molecule linked to the 5′-position of thenucleoside.
 50. The compound of claim 49 wherein at least one of saidnucleosides is a 2′-deoxy-2′-fluoro or a 2′-methoxy nucleoside.